The Glutamate Cascade: Common Pathways of Central Nervous System Disease States

United States

Objective

To explore the evidence that the "glutamate cascade" appears to be associated with several seemingly diverse disease processes of the CNS. The related excitotoxic cascade has been demonstrated in some of these processes. The hope is to stimulate a cross-fertilization of ideas in basic brain mechanisms and preclinical and clinical medications development of direct or indirect glutamatergic antagonists in diverse medical disciplines which focus on these disorders.

Disorders in which a role for glutamate have been demonstrated include: addiction (tolerance, sensitization, dependence, and/or neurotoxicity that is associated with drugs of abuse including opiates, cocaine, amphetamine, alcohol); stroke; epilepsy; brain trauma; certain types of neuropathic pain; AIDS dementia; schizophrenia; depression; anxiety; and aging/Alzheimer's. A role of NMDA receptor complex and NO has also been implicated in LTP and in other models for learning and memory.

NIH Institutions Comprising Agenda Committee: NIDA, NINDS, NIAAA, NIMH, NIA, NIDDK, NICHD, NIDR, NIAID

Speaker Abstracts

The Role df Glutamate in Chronic Inflammatory Pain and Painful Peripheral Neuropathies

Gary J. Bennett, Ph.D.
Allegheny University of the Health Sciences

Somatosensory primary afferent axons that respond specifically to tissue-damaging stimuli (nociceptors) use glutamate (and aspartate) as a neurotransmitter. Neurons in the spinal cord dorsal horn that respond to nociceptor input express all three subtypes of glutamate receptor.

In inflammation, nociceptors with unmyelinated axons (C-nociceptors) are sensitized such that they discharge spontaneously, respond to normally innocuous stimuli, and have a supernormal response to noxious stimuli. Nociceptor sensitization underlies the spontaneous pain and hypersensitivity of the injured site. The area surrounding the injured site also becomes a source of pain and hypersensitivity, and this is now known to be due, at least in part, to an NMDA receptor-mediated hyperexcitability in spinal neurons evoked by C-nociceptor input from the injured site. NMDA receptor blockade reduces the pain of inflammation, but has little or no effect on an acute pain stimulus. The chronic pain syndromes due to peripheral neuropathies are now believed to be related, in part, to a similar NMDA receptor-mediated hyperexcitability in spinal neurons. Axotomized C-nociceptors begin to discharge ectopically, and this has the same effect as the discharge of sensitized C-nociceptors it evokes NMDA receptor-mediated central hyperexcitability. NMDA receptor antagonists block neuropathic pain in animals, and early clinical trials suggest that they are effective drugs for human painful peripheral neuropathies. High levels of activity at NMDA receptors in the brain are known to lead to neuronal death or dysfunction. There is evidence that pain-evoked glutamate release is excitotoxic to small, presumed inhibitory interneurons, in the spinal dorsal horn.

Manipulating Glutamatergic Neurotransmission for Therapeutic Gain: The Example of Brain Ischemia

Dennis W. Choi, M.D., Ph.D.
Washington University School of Medicine

Since most fast excitatory synaptic neurotransmission in the mammalian brain is mediated by glutamate, it is plausible that many neurological or psychiatric disorders may be associated with abnormalities in the glutamatergic signaling system, and that therapeutic benefits might be attainable through the manipulation of this system. A prominent example of both the potential benefits, and the complex pitfalls, associated with the therapeutic manipulation of the glutamate system can be found in the setting of ischemic brain injury. The excitotoxic overactivation of glutamate receptors likely contributes directly to the neuronal and glial cell loss induced by ischemia, to a large extent reflecting excess Na+ and Ca2+ influx and, at least in the case of neurons, consequent cell necrosis. However, efforts to administer either calcium channel blockers, or pharmacological N-methyl-D-aspartate (NMDA) receptor antagonists acutely to patients suffering focal brain ischemia have so far been disappointing. The latter drugs have produced substantial side effects, and neither class of drugs has exhibited clear protective benefits. While the side effects of glutamate antagonists may be surmountable through improving drug selectivity at the level of receptor subtypes or spatial localization, lack of efficacy may point to a flaw at the level of underlying assumptions. Perhaps in man, reduction of ischemic excitotoxicity will require block of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors in addition to block of NMDA receptors. Another possible problem may be lie in the growing body of evidence that suggests that ischemia may trigger substantial amounts of programmed cell death, culminating in apoptosis, in addition to excitotoxic necrosis. One can wonder whether reduction in intracellular free calcium induced by the pharmacological blockade of glutamate receptors or even voltage-gated calcium channels might enhance brain cell susceptibility to apoptosis enough to negate the benefits associated with attenuating excitotoxic necrosis.

Glutamatergic Mechanisms in the Cause and Treatment of Parkinson's Disease

J. Timothy Greenamyre, M.D., Ph.D.
Emory University

The glutamatergic system may be important in the pathogenesis and the pathophysiology of Parkinson's disease (PD). A defect in mitochondrial energy metabolism has been found in PD, and we and others have demonstrated that mitochondrial dysfunction can lead to secondary or "weak" excitotoxicity. Metabolic impairment depletes ATP, depresses Na+/K+-ATPase activity and causes graded neuronal depolarization. This relieves the voltage-dependent Mg++ block of the NMDA receptor and allows innocuous levels of glutamate to become lethal. Mitochondrial impairment also disrupts cellular calcium homeostasis. Increased activation of the NMDA receptor, in turn, leads to further mitochondrial impairment and damage. In large part, this occurs because calcium entering a neuron through NMDA receptors has "privileged" access to mitochondria where it leads to free radical production and mitochondrial depolarization. Thus, there may be a feed-forward cycle wherein mitochondrial dysfunction causes NMDA receptor activation which leads to further mitochondrial impairment. In this scenario, NMDA receptor antagonists may be neuroprotective.

Once PD is established, it is clear that several glutamatergic pathways, including the corticostriatal and subthalamofugal projections, become overactive. This causes regulatory changes in basal ganglia glutamate receptor subunits. Moreover, it suggests that glutamate receptor antagonists may be useful as antiparkinsonian medications. We have shown in MPTP-treated parkinsonian monkeys that NMDA and non-NMDA antagonists have antiparkinsonian efficacy. They may also suppress the involuntary movements that are associated with chronic dopaminergic therapy. Several clinical trials are now underway to test whether manipulation of the glutamatergic system has beneficial effects in PD. Initial results should be available by the end of the year. (Supported by NS33779, AG11755, AG14648 and the Huntington's Disease Society of America.)

Kainate Receptor Deficient Mice

C. Mulle; A. Sailer; I. Perez-Otano; H. Dickinson-Anson; P. Castillo; I. Bureau; C. Maron; F.H. Gage; J. Mann; B. Bettler; and Stephen F. Heinemann, Ph.D.^1
1The Salk Institute

Pharmacological studies in the mammalian brain have revealed three classes of glutamate activated ligand gated receptor channels, AMPA, kainate and NMDA. Because of a lack of specific drugs, it has been difficult to separate synaptic currents mediated by AMPA and kainate receptors. Thus, the role of the kainate receptor system in the brain has not been studied intensively and remains a mystery. We have used a genetic approach to define the function of kainate receptors in the mouse brain. Mutations in the kainate receptor genes have been made in mice in order to elucidate the function of the kainate receptor system in the brain. We have produced a null mutation in the mouse GluR 6 kainate receptor subunit. The homozygous GluR 6 "knock-out" mouse is viable and healthy, although there is about a 10 percent reduction in weight. Both GluR 6 subunit specific RNA and GluR 6 subunit protein are not detected in the mutant mouse brain. Kainate binding is absent in areas of the brain which normally have high levels of GluR 6 specific RNA, i.e. the CA3 region and the dentate gyrus in the hippocampus. Analysis of synaptic transmission at the mossy fiber- CA3 synapse demonstrates that kainate receptors mediate a post-synaptic current which is absent in the GluR6 "knock-out" mouse. Behavioral analysis of the GluR6 "knock-out" mouse showed that they exhibit reduced locomotion activity but that they can learn motor and water maze tasks. The homozygous GluR 6 "knock-out" mouse is resistant to kainate induced seizures as assayed by seizure onset and immediate early gene activation in the hippocampus. These results prove that the kainate receptor subunit GluR 6 is a subunit of a kainate receptor which mediates a post-synaptic current during synaptic transmission in the hippocampus as well as the epileptogenic effects of kainate, a model of human temporal lobe epilepsy. This observation suggests that drugs which target the GluR 6 subunit may have therapeutic value for treatment of epilepsy.

Alcohol and NMDA Receptor Function

Paula L. Hoffman, Ph.D.
University of Colorado Health Sciences Center

Physical dependence on alcohol, which is one component of the alcohol dependence syndrome, is thought to result from an adaptation of the CNS to the chronic presence of alcohol, and is defined by the appearance of withdrawal signs and symptoms, including seizures (convulsions), upon cessation of alcohol intake. Neuronal systems that undergo adaptation to chronic ethanol exposure may be those that are sensitive to acute perturbation by ethanol. Acutely, ethanol is a potent inhibitor of the function of the N-methyl-D-aspartate (NMDA) subtype of glutamate receptor. Following chronic exposure of animals to alcohol, there is evidence for an up-regulation of NMDA receptors, and/or a change in NMDA receptor subunit composition, in several brain areas. The time course of this up-regulation parallels the time course of alcohol withdrawal convulsions, and these convulsions can be attenuated by NMDA receptor antagonists. Prevention of the up-regulation of the NMDA receptor by ganglioside treatment also reduces withdrawal seizures, supporting the role of changes in the NMDA receptor in alcohol withdrawal/physical dependence. NMDA receptor function is also increased in neuronal cultures exposed chronically to ethanol in vitro, and these ethanol-exposed/withdrawn neurons show enhanced susceptibility to glutamate-induced excitotoxic death. This toxicity can be blocked with NMDA receptor antagonists, but not with diazepam. Withdrawal-induced glutamate excitotoxicity may underlie the neuronal damage observed in chronic alcoholics. These findings suggest the possibility of development of therapeutic agents to treat both alcohol withdrawal seizures and withdrawal-induced neuronal damage. (Supported by NIAAA and the Banbury Foundation.)

Role of Glutamate Receptor Maturation in Perinatal Brain Injury

Frances E. Jensen, M.D.
Children's Hospital (Boston) and Harvard Medical School

Glutamate receptor expression is developmentally regulated and data suggest that this is likely to contribute to age-dependent differences in vulnerability to hypoxic/ischemia and seizure-induced neuronal death. Glutamate neurotransmission is generally enhanced in the immature brain at ages when certain glutamate receptors are transiently overexpressed and/or receptor subunit composition differs compared to the adult. Glutamate receptors play a critical role in neuronal plasticity and activity mediated growth during brain development. However, the immature brain appears to be more vulnerable than the adult to certain forms of excitotoxic neuronal injury, suggesting that the maturational state of glutamate receptors modifies response to injury. Furthermore, clinical data demonstrate that seizures are most common in the neonatal brain, and animal models suggest that this heightened excitability correlates with transiently enhanced glutamate receptor activity. We have shown that glutamate receptor expression can be modified by brain injury during early development. Both hypoxia and seizures alter glutamate receptor subunit expression and in some cases chronically alter neuronal excitability. Given the maturational differences in excitatory amino acid mediated injury, age specific therapeutic strategies represent an important future research direction. The immature brain represents a unique challenge because therapeutic strategies will have to be devised that minimize adverse effects on physiological roles of the glutamate receptor in plasticity and synaptic development.

Excitotoxic Apoptosis and Necrosis in AIDS Dementia and Stroke: Potential Treatment With NMDA Open-Channel Blockers and Nitric Oxide-Related Species

Stuart A. Lipton, M.D., Ph.D.
Brigham and Women's Hospital
Harvard Medical School

Neuronal injury in a number of neurodegenerative disorders, including AIDS dementia and stroke, is mediated by excitotoxic pathways leading to free radical generation. Apoptosis results if the initial insult is relatively mild, but necrosis intervenes if the initial insult is fulminant [ref. 1-6]. How can HIV-1 result in neuronal damage if the predominant cell type infected in the CNS is the macrophage/microglia? Experiments from several laboratories have lent support for the existence of HIV- and immune-related excitotoxins secreted by activated microglia and astrocytes. These substances may include arachidonic acid, platelet-activating factor, free radicals (NO. and O2.), glutamate, quinolinate, cysteine, amines, cytokines (TNF-, IL1-, IL-6), and as yet unidentified factors (Fig. 1). A final common pathway for neuronal susceptibility appears to underlie AIDS dementia, similar in many ways to that observed in focal cerebral ischemia or stroke. This mechanism involves overactivation of glutamate receptors, predominantly the N-methyl-d-aspartate (NMDA) subtype, followed by excessive influx of calcium and the generation of free radicals. After outlining these pathways, this lecture will discuss the development of clinically-tolerated NMDA antagonists, including (i) the open-channel blocker memantine, and (ii) redox-active, NO-related species such as nitroglycerin (see Fig. 2) [ref. 7].

References

1. Ankarcrona, M.; Dypbukt, J.M.; Bonfoco, E.; Zhivotovsky, B.; Orrenius, S.; Lipton, S.A.; and Nicotera, P. Glutamate-induced neuronal death: A succession of necrosis or apoptosis depending on mitochondrial function. Neuron 15:961-973, 1995.
2. Bonfoco, E.; Krainc, D.; Ankarcrona, M.; Nicotera, P.; and Lipton, S.A. Apoptosis and necrosis: Two distinct events induced respectively by mild and intense insults with NMDA or nitric oxide/superoxide in cortical cell cultures. Proc Natl Acad Sci USA 92:7162-7166, 1995.
3. Lipton, S.A. Neurobiology: HIV displays its coat of arms. Nature 367:13-114, 1994.
4. Lipton, S.A.; Choi, Y.-B.; Pan, Z.-H.; Lei, S.Z.; Chen, H.-S.V.; Sucher, N.J.; Loscalzo, J.; Singel, D.J.; and Stamler, J.S. A redox-based mechanism for the neuroprotective and neurodestructive effects of nitric oxide and related nitroso-compounds. Nature 364:626-632, 1993.
5. Lipton, S.A., and Gendelman, H.E. The dementia associated with the acquired immunodeficiency syndrome. N Engl J Med 332:934-40, 1995.
6. Lipton, S.A., and Rosenberg, P.A. Mechanisms of disease: Excitatory amino acids as a final common pathway for neurologic disorders. N Engl J Med 330:613-622, 1994.
7. Das, S.; Sasaki, Y.F.; Rothe, T.; Premkumar, L.S.; Takasu, M.; Crandall, J.E.; Dikkes, P.; Connor, D.A.; Rayudu, P.V.; Cheung, W.; Chen, H.-S.V.; Lipton, S.A.; and Nakanishi, N. Increased NMDA current and spine density in mice lacking the NMDAR subunit, NR3A. Nature, in press.

Effect of Glutamate Blockers on Human Pain

Mitchell B. Max, M.D.
National Institute of Dental Research
National Institutes of Health

Animal studies have shown injury can sensitize spinal sensory neurons and cause prolonged and severe pain through excitation mediated via glutamate receptors. N-methyl-D-aspartate (NMDA) receptors are the subclass of glutamate receptors that have been most extensively studied. Although ketamine reduces a number of types of experimental and clinical pain in humans, the drug also causes severe cognitive and affective side effects, making it unattractive for chronic treatment. We have carried out clinical trials of the orally available NMDA receptor antagonist and anti-tussive, dextromethorphan in patients with painful diabetic neuropathy and postherpetic neuralgia. Dextromethorphan, 400 mg/day, reduced pain in 32 patients with diabetic neuropathy by about 25 percent relative to placebo, but had little effect on post-herpetic neuralgia.

Non-NMDA glutamate receptors may also be involved in pain and hyperalgesia. We obtained LY293558 from Eli Lilly to assess its analgesic and anti-hyperalgesic effects in human volunteers. This was the first administration of this compound or any AMPA/kainate blocker to humans, so we carried out extensive Phase 1 safety testing as well as pain studies in 26 human volunteers. During rising-dose studies in each subject, we assessed responses to brief painful electrical and heat stimuli administered to undamaged skin. After the highest dose not producing significant side effects (maximal tolerated dose, MTD) was determined for each subject, he or she entered a 3-session, randomized, double-blind crossover study comparing the effects of the MTD, 1/3 MTD, or placebo on capsaicin-evoked pain, and areas of mechanical hyperalgesia and allodynia. There was a dose-related reduction of pain, hyperalgesia, and allodynia.Allodynia and hyperalgesia were reduced by about half by the 1/3 MTD, a dose that had no greater side effects than placebo. This promising result led Eli Lilly to carry out further studies with superselective compounds, which suggested that the anti-hyperalgesic effect was due to blockade of GluR5 kainate-type receptors, while sedative effects result from GluR1-4 AMPA receptors. If current studies of LY 293558 in patients with clinical pain confirm these results, kainate antagonists may offer a powerful and nontoxic treatment for pain.

Glutamate Receptor Plasticity in the Hippocampus: Implications for Aging

John H. Morrison, Ph.D.
Fishberg Research Center for Neurobiology
Mount Sinai School of Medicine

Recent immunohistochemical studies have demonstrated both a high degree of specificity in hippocampal circuits with respect to representation of glutamate receptor families and subunits, as well as a surprising degree of glutamate receptor plasticity in response to aging, deafferentation, and neuroendocrine status. We have hypothesized that such glutamate receptor plasticity underlies age-related functional impairment, as well as modifications in excitatory transmission and morphology that can be related to circulating estrogen levels. Three examples of such plasticity will be presented: 1) A subunit and circuit-specific decrease in NMDAR1 in the perforant path terminal zone of the dentate gyrus molecular layer in aged primates; 2) An increase in NMDAR1 protein and mRNA in the dentate gyrus molecular layer following perforant path lesions in rats; and 3) an estrogen-induced increase in NMDAR1 protein, but not mRNA, in CA1 and dentate gyrus following ovariectomy. These data will be discussed in the context of age-related memory impairment in the absence of neuron loss, as well as their relevance to neurodegenerative disorders such as Alzheimer's disease.

The NMDA/Nitric Oxide Synthase Cascade and Opioid Analgesia and Tolerance

Gavril W. Pasternak, M.D., Ph.D.
Cornell University Medical College

The NMDA receptor has been implicated in a wide variety of neurological functions, acting in many cases through the activation of neuronal nitric oxide synthase (nNOS). Work from a number of laboratories has implicated NMDA receptors in the production of tolerance to opioid analgesics, as shown by the prevention of morphine tolerance by noncompetitive NMDA antagonists such as MK801. Subsequent work has now extended these observations to various other classes of NMDA antagonists, including competitive antagonists and glycine-site antagonists. Tolerance to delta drugs also is sensitive to these same agents. The loss of analgesic potency by delta drugs with repeated administration is prevented by these NMDA antaognists. Kappa analgesics, however, yield a different picture. In our studies, we have not observed an effect of NMDA agents on either kappa1 or kappa3 tolerance, although there are some suggestive reports in the literature. In many situations, stimulation of NMDA receptors leads to the activation of neuronal nitric oxide synthase (nNOS). Inhibition of this enzyme also interferes with the production of mu and delta tolerance, without altering kappa tolerance. Many splice variants of nNOS exist, raising the question of whether the role of nNOS in morphine tolerance may be limited to a single isoform. Using antisense approaches, we have selectively downregulated the major form of nNOS and a minor nNOS splice variant and looked at their pharmacology. The predominant nNOS isoform is important in the development of morphine tolerance, resulting in a loss of morphine analgesic activity. However, the minor nNOS variant has an opposite action. Unlike the major isoform, the minor isoform facilitates morphine analgesia. These observations illustrate the complexity of the NMDA/nNOS system in pain perception and opioid tolerance.

NMDA Antagonists Modify Opiate Drug-Seeking: Preclinical and Preliminary Clinical Findings

Piotr Popik, M.D., Ph.D.
Institute of Pharmacology
Polish Academy of Sciences, Poland

Traditional "anti-addictive" pharmacotherapies are "drug-targeted," despite the fact that all of the drugs of abuse produce the same reinforcing (rewarding) effects and their long term administration results in drug addiction. However, the same inhibitory actions of N-methyl-D-aspartate (NMDA) receptor antagonists on phenomena related to drug seeking behavior produced by all addictive substances suggest that glutamatergic receptors may be a final common pathway to all addictions and a possible therapeutic target. Presented are the data illustrating inhibitory effects of NMDA receptor antagonists on the expression and maintenance of physical and motivational aspects of morphine dependence in rats and mice. Next, the data concerning inhibitory effects of several (uncompetitive: memantine, glycine/NMDA: L-701,324 and competitive: NPC 17742) NMDA receptor antagonists on the acquisition, expression and extinction of morphine-induced conditioned reward are discussed. Brain areas sensitive to the treatment with NMDA receptor antagonists have been identified as the administration of NPC 17742 into the nucleus accumbens and ventral tegmental area produced the same, inhibitory effects on the expression of morphine-conditioned reward. These preclinical data are followed by presentation of preliminary clinical findings suggesting that the uncompetitive NMDA antagonist, dextromethorphan may facilitate detoxification from heroin and inhibit craving for this drug in human addicts. Despite the "bad reputation" of some NMDA receptor antagonists that produce ataxia, memory disturbances and psychotomimetic effects, some low affinity, highly voltage dependent uncompetitive antagonists like memantine and dextromethorphan as well as glycine/NMDA site antagonists may have beneficial effects on drug dependence and addiction.

Glutamate During the Natural History of Psychostimulant Dependence: Synaptic Plasticity and Medications Development

Luigi Pulvirenti, M.D.
University of Rome - Tor Vergata, Italy

The natural history of drug dependence is characterized by phases of acquisition, maintenance, extinction and relapse of drug taking. All these phases have now been successfully mimicked using a rodent model of intravenous drug self-administration and the psychostimulant addiction cycle has been extensively investigated. Much evidence suggests that glutamate may play a critical role. During the first days of acquisition of cocaine-seeking behavior a glutamate-dependent enhancement of synaptic efficacy was found using evoked field responses in the rat nucleus accumbens, a critical neural substrate mediating cocaine self-administration. Furthermore, intact glutamatergic neurotransmission was shown to be essential for the full expression of the reinforcing properties of cocaine during the maintenance phase of cocaine self-administration. Later phases of cocaine dependence were also found to be affected by indirect glutamatergic manipulation. Inhibition of nitric oxide synthesis reduced the reinforcing properties of cocaine, cocaine craving during the extinction phase and later relapse into cocaine-seeking behavior. Therefore, a potential for medication development targeting glutamate neurotransmission exists and is further supported by preclinical observations showing that dextromethorphan, a clinically safe medication with glutamate antagonistic properties, reduced the absolute reinforcing properties of cocaine. Glutamate-dependent changes of synaptic efficacy within critical limbic circuits may therefore be part of the early events of the addiction cycle leading to the development of abuse and part of the long-lasting behavioral changes of the natural history of dependence, warranting further studies on pharmacological intervention at the glutamate level for the therapy of drug abuse.

Glutamate Neurotransmission in Epilepsy: New Treatment Strategies

Michael A. Rogawski, M.D., Ph.D.
National Institute of Neurological Disorders and Stroke
National Institutes of Health

Several lines of evidence establish the importance of glutamate-mediated neurotransmission in epilepsy and epileptogenesis. (i) Massive release of glutamate has been demonstrated in human brain during seizures. (ii) Glutamate and other agonists of glutamate receptors are powerful convulsants. (iii) Manipulations that enhance glutamate release or potentiate glutamate receptor function can result in epileptiform activity and seizures. (iv) Modification of glutamate receptor function in transgenic animals can lead to the development of epilepsy. Most importantly, however, antagonists of glutamate receptors have broad spectrum antiseizure activity in in vitro and in vivo model systems. The possible use of NMDA receptor antagonists in the treatment of seizures has been extensively investigated, but results to date have not been encouraging mainly because the NMDA receptor antagonists evaluated have had significant toxicities. There are a variety of novel approaches currently under investigation that may lead to NMDA receptor antagonists with improved side effect profiles, including selective antagonists of NMDA receptors containing the NR2B subunit, glycine site antagonists and low affinity channel blockers. Recently, selective antagonists of non-NMDA (AMPA/kainate) receptors have become available and provide a new direction for the development of epilepsy therapies. Potential approaches include 2,3-benzodiazepine selective allosteric AMPA antagonists, modulators of AMPA receptor desensitization and antagonists of Ca2+-permeable AMPA receptors (i.e., those lacking the GluR2 subunit). Recent studies have suggested that kainate receptors may be particularly promising targets for epilepsy therapy as they seem to play a dual role as inhibitors of GABA release as well as in mediating postsynaptic excitation. Emerging work on kainate receptor-selective antagonists has focused on drugs that target kainate receptors containing the GluR5 subunit.

Is Modulation of Glutamatergic Transmission a Viable Approach for Developing Novel Anxiolytics?

Phil Skolnick, Ph.D.
Lilly Research Laboratories

Most currently prescribed antianxiety agents act by enhancing GABAergic neurotransmission. Moreover, converging lines of evidence suggest GABAergic dysfunction can contribute to the pathophysiology of anxiety. Nonetheless, both anxiolytic and anxiogenic actions can be produced by agents that affect other neurotransmitter systems indicating that like other psychiatric disorders, anxiety (and anxiolysis) is not mediated by a single neurotransmitter system. It has been proposed that disruption of the homeostatic balance between fast-acting, ligand-gated ion channels utilizing glutamate and GABA as transmitters can result in the development and expression of anxiety. Strategies that reduce transmission at the N-methyl-D-aspartate (NMDA) subtypes of glutamate receptor are effective in preclinical tests that predict anxiolytic activity in humans. For example, functional NMDA antagonists acting at the multiple, allosterically linked sites on this family of ligand-gated ion channels mimic the effects of clinically active agents in the elevated plus maze, ultrasonic vocalization, potentiated startle, and thirsty rat (Vogel's) conflict tests. Moreover, compounds that reduce glutamate release via presynaptic metabotropic glutamate receptors exhibit a similar, but not identical spectrum of activity. I will discuss the potential pitfalls and advantages of achieving an anxiolytic action through modulating glutamatergic transmission.

Glutamate-Mediated Neuroplasticity: Role in Amphetamine Addiction

Marina E. Wolf, Ph.D.
The Chicago Medical School

Work from our laboratory and others has demonstrated that the development of sensitization requires glutamate transmission, suggesting mechanistic similarities to other forms of neuronal plasticity. More recently, we have shown that sensitization involves adaptations in glutamate transmission in the mesoaccumbens DA system. After repeated amphetamine, VTA DA cells are supersensitive to the excitatory effects of glutamate and AMPA. This is transient, present after 3 but not 14 days of withdrawal, and does not reflect increased expression of AMPA receptor subunits (measured at protein or mRNA levels). NAc neurons show subsensitivity to glutamate, NMDA and AMPA at both withdrawals. Decreased levels of mRNA and immunoreactivity for GluR1, GluR2 and NR1 subunits were found in NAc at the 14 day withdrawal time, which may in part explain the electrophysiological findings. We also examined PFC, a major source of glutamate projections to VTA. After 3 days' withdrawal, we found increased GluR1 expression and increased sensitivity of PFC cells to glutamate. This may contribute to transient increases in excitatory drive to VTA DA cells thought to mediate induction of sensitization. Increases in excitatory drive may also reflect effects of amphetamine within the VTA, since microdialysis studies have found a delayed increase in glutamate efflux after systemic or local amphetamine. This is prevented by agents that also prevent sensitization (MK-801 and SCH 23390), suggesting a link between increased glutamate efflux and induction. The link could reflect LTP-like mechanisms or mild excitotoxic damage to DA neurons, either of which could result in transient hyperexcitability. (Supported by DA09621.)

Acamprosate, a Novel Anti-Craving Compound Acts Via Glutamatergic Pathways

Walter Zieglgänsberger, M.D., Ph.D.
Max Planck - Institute of Psychiatry, Munich, Germany

Acamprosate (calcium acetyl-homotaurinate) shows promise for treatment of alcoholism and is now registered for clinical use in the European Community. It reduces alcohol intake when administered to rodents and human alcoholics (Sass et.al 1996). This compound reduces the marked increase in expression of immediate-early-genes during alcohol withdrawal in a variety of brain regions. Acamprosate by itself increases expression of c-fos e.g. in the hippocampus (Putzke et al. 1997) and increases the expression of certain splice variants (e.g. NMDAR1-4 in the hippocampus) of the NMDAR1 (Putzke et al. in press). Acamprosate alters postsynaptic efficacy of excitatory amino acids: it attenuates postsynaptic activity of EAA agonists in neocortical neurons and enhances NMDA receptor mediated synaptic transmission (Madamba et al. 1997; Berton et al. 1998) in hippocampal and nucl. accumbens neurons. Acamprosate does not enhance Cl-currents following the activation of GABAA receptors in various preparations. In HEK 293 cells co-expressing NMDAR1 and NMDAR2A subunits nanomolar concentrations of acamprosate dose-dependently and reversibly reduced the NMDA-induced ion current.
There is no indication from the current assessment of humans of any psychotropic side effects of acamprosate or any potential for abuse or dependence. Animals will self-administer MK-801, but not acamprosate, and cannot be trained to discriminate subjective effects of acamprosate or to substitute acamprosate for alcohol in a drug discrimination paradigm. Acamprosate has been shown to be devoid of hypnotic, anxiolytic or muscle relaxant properties. There is no evidence of any antidepressant or other psychotropic effect of acamprosate (see Spanagel and Zieglgänsberger, TIPS 18, 1997).

Schizophrenia: A Glutamatergic Perspective

Stephen R. Zukin, M.D.
National Institute on Drug Abuse
National Institutes of Health

In the late 1950s the ability of the dissociative anesthetic drug phencyclidine (1-(1-phenylcyclohexyl) piperidine; PCP) to elicit a prolonged psychotic state in surgical patients was recognized. Experimental studies then revealed the ability of single small doses of PCP to elicit brief schizophrenia-like symptoms and cognitive abnormalities in normal volunteers. Similar experiments in previously stabilized chronic schizophrenic subjects led to prompt rekindling and exacerbation of disease-specific symptoms persisting up to many weeks. In the 1970s PCP became a prominent drug of abuse; in many cases patients with PCP-induced psychotic reactions could not be clinically distinguished from patients with naturally-occurring schizophrenia. In 1979 a unique brain binding site, selective for drugs with PCP-like behavioral properties, was discovered and characterized. In the 1980s whole-animal electrophysiological evidence demonstrated that PCP-like drugs were potent antagonists of the N-methyl-D-aspartate (NMDA) class of glutamate receptors. This interaction was confirmed at the cellular and biochemical levels. Characterization of the mode of activation of the NMDA receptor complex revealed multiple regulatory and modulatory sites, including a PCP binding site located within the ligand-gated ionophore, and a non-strychnine-sensitive site at which glycine served as an obligatory co-agonist, the local glycine concentration determining the degree of agonist-induced channel activation. Several lines of evidence support the hypothesis that NMDA receptor-mediated neurotransmission is abnormal in schizophrenia. These include observations that 1) large oral doses of glycine diminish negative-symptom ratings in neuroleptic-treated schizophrenic patients; 2) neuroanatomical abnormalities reported in schizophrenia correspond to areas rich in NMDA receptors; 3) PCP-like drugs induce abnormalities in cortical evoked potentials similar to those seen in schizophrenia; 4) neuropsychological abnormalities in schizophrenia involve glutamatergic mechanisms; and 5) a variety of additional neurochemical, neuroanatomical and physiological findings.

Poster Abstracts

Glutamate-Dopamine Interactions In Humans (GII-B)

Abi-Saab, W.M.; D'Souza, D.C.; Karper, L.P.; Madonick, S.; Belger, A.; Gil, R.; Bennett, A.; Abi-Dargham, A.; Heninger, G.R.; Charney, D.S.; and Krystal, J.H.

The interactions of glutamate and dopamine systems have been implicated in the pathophysiology of several neuropsychiatric and substance abuse disorders. To study these interactions, we are examining the effects of dopamine agonists and antagonists on subhypnotic doses of the N-methyl D-aspartate (NMDA) glutamate receptor antagonist, ketamine, in humans.

Methods

The two studies employed a randomly balanced, placebo-controlled design where in the first study healthy subjects (n=20) received haloperidol 5 mg or placebo 2 hours prior to ketamine (0.26 mg/kg bolus then 0.65 mg/kg over 1 hour) or placebo; or in the second study, healthy subjects (n=7) received amphetamine (0.25 mg/kg over 1 min) or placebo and ketamine (0.23 mg/kg bolus then 0.5 mg/kg over 1 hour) or placebo.

Results

Study 1: Ketamine produced psychosis, perceptual alterations, and performance decrements in abstraction and Wisconsin Card Sorting Test (WCST). While haloperidol did not alter psychosis, perceptual changes, or euphoria produced by ketamine, it was effective in reducing the abstraction and WCST impairments. Study 2: Our preliminary data suggest that amphetamine had no effect on the ketamine-induced psychosis or euphoria. However, it may have reduced attention deficits produced by ketamine.

Implications

First, the impairments in executive cognitive functions arising from deficits in NMDA receptor function appear to be mediated in part by excessive stimulation of dopamine-2 receptors. Second, neither the psychosis nor the euphoria produced by NMDA antagonists appear dependent upon D2 receptor stimulation. Third, the failure of amphetamine to exacerbate the ketamine psychosis may suggest that the NMDA receptor deficit model is most relevant to a subset of schizophrenic patients with poor responses to typical neuroleptics, since neuroleptic responsive patients tend to show worsening of psychosis during amphetamine infusion.

Lamotrigine Effect on Ketamine Induced Neuropsychiatric Symptoms in Humans (GII-B)

Anand, A.; Charney, D.S.; Cappiello, A.; Berman, R.M.; Oren, D.A.; and Krystal, J.H.

Ketamine is a non-competitive antagonist of the NMDA receptor that produces psychosis, perceptual alterations, thought disorder, and mood changes in healthy subjects. Preclinical studies suggest that ketamine may increase cortical glutamate levels. As a result, we evaluated the capacity of lamotrigine, a drug reported to reduce presynaptic glutamate release, to reduce ketamine effects in humans.

Methods

In an ongoing study, healthy subjects (n = 14) completed 4 test days involving the administration of lamotrigine 300 mg, p.o. or placebo 2 hours prior to ketamine (0.23 mg/kg, i.v. bolus and 0.5 mg/kg/hr i.v. infusion) or placebo in a randomized order under double blind conditions. Cognitive, behavioral and neuroendocrine assessments were performed.

Results

Preliminary analysis suggests that lamotrigine blunted the amnestic effects of ketamine, as assessed by the Hopkins Verbal Learning Test (HVLT) (p < 0.05), the perceptual effects of ketamine as assessed by the Clinician-Administered Dissociative States Scale (CADSS) (p < 0.0001), psychiatric symptoms as assessed by the total Brief Psychiatric Rating Scale (BPRS) (p < 0.03) and negative symptoms as assessed by the key negative symptoms sub-scale of the BPRS (p < 0.05). There was a trend for a decrease in the positive symptoms sub-scale of the BPRS (p < 0.07). Ketamine induced a significant mood elevation measured by the Young Mania Rating Scale (YMRS) (p < 0.001). Lamotrigine did not decrease ketamine induced mood elevation. In 8/14 subjects lamotrigine enhanced the mood elevating effects of ketamine.

Significance

These preliminary data provide indirect support for the hypothesis that the psychotomimetic effects of ketamine are mediated by enhancement of glutamate release. Lamotrigine may be useful in the treatment of schizophrenia, depression, and dissociative disorders.

Is Ibogaine Like MK-801? Evaluation of in Vivo Neurobiological Evidence (GI-B)

Baumann, M.H.; Rothman, R.B.; and Ali, S.F.

Ibogaine (IBO) is a naturally-occurring indole compound that is being evaluated as a potential medication for substance use disorders. Although the precise mechanism of IBO action is unclear, recent in vitro data show this drug displays properties similar to the non-competitive NMDA antagonist MK-801. The purpose of the present work was to compare in vivo neurobiological effects of IBO and MK-801 in rats. Groups of male rats (N=6-8/group) were decapitated 30 and 60 min after receiving ip IBO (10 & 100 mg/kg), MK-801 (0.1 & 1.0 mg/kg) or vehicle. Trunk blood was collected for the analysis of plasma prolactin and corticosterone; brains were harvested and dissected for determination of DA, 5-HT and their metabolites. Both IBO and MK-801 increased corticosterone secretion, but only IBO elevated plasma prolactin. IBO produced dramatic reductions in tissue DA levels with a concurrent increases in the metabolites, DOPAC and HVA. This profile of IBO-induced changes in DA transmission was observed in the striatum, olfactory tubercle, and hypothalamus. The effects of MK-801 on DA metabolism did not mimic IBO, as MK-801 tended to increase DA and its metabolites. Neither drug appreciably affected 5-HT systems. Our results suggest that the effects of IBO on neuroendocrine function and DA transmission are not due to MK-801-like properties of IBO. Thus, the in vivo mechanism of IBO action can not be explained simply on the basis of antagonism at NMDA receptors.

NMDA Receptor Function in Major Depression (GII-B)

Cappiello, A.; Berman, R.M.; Anand, A.; Charney, D.S.; and Krystal, J.H.

A large body of preclinical research supports the hypothesis that the glutamate system is involved in the mechanism of action of antidepressants, potentially playing a role in the final common pathway. The following study provides preliminary insights on the role of NMDA function in mediating symptoms of major depression.

Methods

Seven subjects (3 female, 4 males), aged 31 to 55, who met DSM-IV criteria for major depression and 18 healthy subjects, aged 18 to 44 underwent two testing conditions in a balanced, double-blinded manner: following an overnight fast, subjects received intravenous administration of ketamine (0.5 mg/kg) or saline administered over 40 minutes. Clinical ratings of depression, psychosis, dissociative state, and cognitive functioning were assessed serially.

Results

Compared to control subjects, depressed subjects experienced significant ketamine-induced changes that consisted of: (1) greater "high" scores on the visual analog scale; (2) decreased verbal fluency performance; and (3) increased positive and negative symptoms on the BPRS. Furthermore, depressed subjects reported marked improvement in depressive symptoms 72 hours after active versus sham infusion (i.e., 25-item Hamilton Depression Rating Scale scores decreased 13 points vs. increased 2 points, respectively [f=3,74, df=5,60, p=0.01]).

Conclusion

Further work is needed to clarify time course of these findings. Preliminary results are consonant with an NMDA model of depression and may suggest a role for NMDA receptor-modulating drugs in the treatment of depression.

NMDA-Induced Dopamine Response is Blunted Following Repeated Kappa-Opioid Receptor Agonist Treatment (GI-B)

Britton, W.B.; Thompson, A.C.; and Shippenberg, T.S.

Repeated exposure to cocaine results in an enhancement of its reinforcing and psychomotor stimulant effects, a phenomenon referred to as sensitization. The involvement of glutamatergic neurons in the initiation and long-term expression of sensitization has been documented. We have previously shown that kappa-opioid receptor agonists (k-agonists) block the development of cocaine-induced sensitization. This treatment also prevents cocaine-induced changes in dopamine neurochemistry. The role of glutamate in mediating these effects of k-agonists are unknown. Accordingly, this study sought to examine the influence of k-agonists on NMDA-evoked dopamine release in the striatum. Sprague-Dawley rats were implanted with permanent indwelling guide cannula aim at the dorsal striatum (n=25). Following recovery, rats were treated with the selective k -agonist U-69593 (0.32 mg/kg/day x 5 days, s.c.) or Vehicle (20 percent propylene glycol, 1ml/kg/day x 5 days, s.c.). Microdialysis was conducted three days after the last treatment. Dopamine levels in the dorsal striatum were evaluated following intrastriatal perfusion of increasing concentrations of NMDA (0M, 100M, 1mM, 10mM, 30mM). The results showed that NMDA perfusion increased striatal dopamine levels in a dose-related manner. Prior administration of U69593 did not alter basal DA levels within this brain region. However, U-69593 treatment significantly inhibited the stimulatory effects of NMDA on striatal DA overflow. These data demonstrate that repeated k -agonist treatment produces long-term adaptation in NMDA-receptor mediated systems. It is hypothesized that these alterations may be one mechanism by which kappa-opioid receptor agonists inhibit the development and long-term expression of behavioral sensitization to cocaine.

Chronic Lithium Robustly Protects Cultured CNS Neurons Against Neurotoxicity Induced by Excitotoxins (GII-B)

Nonaka, S.; Hough, C.; and Chuang, D-M.

Lithium is best known for its therapeutic efficacy in the treatment of manic depressive illness. The molecular mechanisms underlying its clinical efficacy remain unclear. To explore the novel actions of lithium, we studied its neuroprotective effects on primary cultures of CNS neurons.

We found that long-term exposure to lithium chloride dramatically protects cultured rat cerebellar, cerebral cortical, and hippocampal neurons against glutamate-induced excitotoxicity which involves apoptosis mediated by N-methyl-D-aspartate (NMDA) receptors. This neuroprotection of lithium is long-lasting, occurs at therapeutically relevant concentrations with an EC50 of approximately 1.3 mM, and requires treatment for 6-7 days for complete protection to occur. In contrast, a 24-hour treatment with lithium was ineffective. The protection in cerebellar neurons is specific for glutamate-induced excitotoxicity and can be attributed to inhibition of NMDA receptor-mediated calcium influx as measured by 45Ca2+ uptake studies and Fura-2 fluorescence microphotometry. The long-term effects of lithium are not due to down-regulation of NMDA receptor subunit proteins and are unlikely related to its known ability to block inositol monophosphatase activity. Our results suggest that modulation of glutamate receptor hyperactivity represents at least part of the molecular mechanisms by which lithium alters brain function and exerts its clinical efficacy in the treatment for manic depressive illness. These novel actions of lithium also suggest that abnormality of glutamatergic neurotransmission as a pathogenic mechanism underlying bipolar illness warrants future investigation.

Two Configurations of Endothelial Glucose Transporter Expression: A Common Mechanism in Human Brain Insult (GII-A)

Cornford, E.M.

Dynamic FDG-PET scans of patients with complex partial seizures demonstrated that the zone of hypometabolism (which includes and extends beyond the anatomical lesion) was also a region of significantly reduced glucose influx. Confirmation of this finding was sought with immunogold electron microscopic quantification of the blood-brain barrier (BBB) Glut1 glucose transporter in tissue from seizure resections resected from patients undergoing surgery for treatment of seizures. Samples were prepared from two different regions of each resection: 1) the most actively spiking epileptogenic site, and 2) the least actively spiking region, as indicated by intraoperative EEG monitoring. Two configurations of endothelial cell Glut1 were observed. About one-half of the capillary profiles examined displayed abundant Glut1 immunoreactivity on both luminal and abluminal endothelial membranes. In the remainder of the profiles, reduced Glut1 labeling was seen; but adjacent erythrocyte membranes remained highly Glut1-immunoreactive, suggesting that reduced endothelial Glut1 reactivity was not attributable to method artifacts. Altered glucose transporter activity in the BBB was characterized by a bimodal Glut1 distribution in which the smaller (Type B) endothelial cells displayed low Glut1 immunoreactivity, while adjacent (and even contiguous) larger (Type A) endothelial cells showed 5-10-fold greater expression of membrane Glut1 transporter protein. Immunogold studies using antisera to human glial fibrillary acidic protein (GFAP) and human serum albumin (HSA) demonstrated increased quantities of these two epitopes in the extravascular regions in which more EEG spiking activity had been demonstrated. These observations indicate that capillary integrity was more compromised, and gliosis was quantitatively increased, in the more actively spiking region of the resection. Altered BBB Glut1 expression has also been observed in brain injury, CNS tumors, and Alzheimer's disease; but not in 8 different normal primate brains examined. In conclusion, common response pathways in CNS disease apparently modify brain glucose acquisition, and emphasize the dynamic contributions of BBB capillaries to human brain homeostasis. (Supported by NS-25554.)

Preliminary Evaluation of the Tolerability/Safety of High Dose Dextromethorphan in Methadone-Maintained Subjects (GI-B)

Cornish, J.W.; Herman, B.H.; Ehrman, R.N.; Childress, A.R.; Bead, V.; Hackett, C.; Caruso, F.S.; Walsh, R.; Martz, K.; Bloch, D.; Oehlert, J.; Hill, J.; O'Brien, C.P.; Bridge, P.; and Vocci, F.

Results of preclinical research suggest that glutamatergic systems play an important role in opioid dependence (cf. Herman & O'Brien Semin Neurosci 9:158-172, 1997). NMDA receptor antagonists (e.g., dextromethorphan (DM)) inhibit and reverse the analgesic effects of morphine in rodents (cf. Inturrisi Sem Neurosci 9:110-119, 1997). Co-administration of glutamatergic antagonists with opioid medications (methadone (M)) may facilitate the dose reduction of these agents and decrease relapse to abused opiates. The present inpatient study evaluated the safety/tolerability of co-administration of DM + M as a precursor to a possible efficacy trial. Fifteen subjects were randomized in a double-blind (DB) fashion to either DM (N = 10) or Placebo (P) (N = 5). In this 16-day (d) study, DM doses (qid) included: 120-, 240-, and 480-mg/d. Daily M doses were between 50-70 mg/d and were constant. Data are presented for the completers (N = 8 for DM; N = 5 for P). Initial analyses focused on the 480 mg/d DM comparisons and included planned comparisons with P counterparts at 1h following medication for: pulse, systolic blood pressure, respiration rate, temperature and affective measures (ARCI subscales) and at 2h for: visual analog scale (VAS) (opiates, nicotine). During the P run-in phase, both groups had similar cardiovascular, pupil size and affective profiles, however both opiate and nicotine VAS showed a two-fold difference. During the DB medication phase, no significant differences between groups were detected on any variable, including euphoria, dysphoria or VAS (opiate, nicotine). The VAS opiate and nicotine effects are of interest. These results suggest that the addition of large doses of DM (up to 480 mg/d) to a moderate dose of M failed to have significant AEs, indicating the apparent tolerability of DM + M. Further studies evaluating DM + M for the treatment of opiate addiction are projected. (Supported by an Interagency Transfer Agreement Between MDD, NIDA and the Philadelphia VA and a CRADA between MDD, NIDA and ALGOS.)

NMDA Receptor Involvement in Anoxia-Induced Disruption of Ionic Homeostasis in Grey Matter In Vitro? (GII-A)

Croning, M.D.R. and Haddad, G.G.

Neuroprotective effects of NMDA receptor antagonists have been demonstrated in animal models of stroke, particularly of the focal ischemia type. However, in brain slices maintained in vitro, subjected to severe hypoxia or anoxia, little, if any, significant reduction is seen in neuronal depolarization or disruption of ionic homeostasis when they are treated with NMDA receptor antagonists.

Therefore we hypothesized that in these slice models, the extracellular acidosis that arises as a result of stimulation of glycolysis during oxygen deprivation may be sufficient to prevent NMDA receptor activation, despite the occurrence of glutamate release and cellular depolarization. In order to test this hypothesis, we subjected rat hippocampal and cortical slices to anoxia with or without concurrent glucose deprivation, and used ion-selective microelectrodes to record alterations in extracellular ion concentrations, monitor the onset of anoxic depolarization, and determined the effect of an NMDA receptor antagonist on these events. We found that reducing glucose availability greatly attenuated the anoxia-induced decrease in extracellular pH both in the CA1 stratum pyramidale of the hippocampus and layers II/III of parietal cortex slices. Under the low glucose, high pH conditions, (Å)-AP-5 (100 M) significantly delayed the onset of anoxic depolarization in both regions. However, it had little effect on the time of onset as a result of anoxia alone. The magnitude of the changes in extracellular [K+] and [Na+] as a result of anoxia Å glucose deprivation were not significantly reduced by (Å)- AP-5, suggesting routes other than the NMDA receptor-ionophore complex, may exist for the fluxes underlying these changes.

Ketamine Exacerbates Symptoms in Schizophrenic Subjects: Evidence for Altered NMDA Receptor Function? (GII-B)

D'Souza, D.C.; Abi-Saab, W.; Gil, R.; Karper, L.; Charney, D.; and Krystal, J.H.

Background

A leading hypothesis suggests that abnormalities in NMDA receptor function contribute to the neurobiology of schizophrenia. It is difficult to study NMDA receptor dysfunction in isolation. The ketamine model provides a direct, clear, interpretable and replicable method to study the consequences of selective deficits in NMDA receptor function and its contributions to the neurobiology of schizophrenia. Ketamine produces schizophrenia-like symptoms in health subjects by noncompetitive NMDA receptor antagonism.

Purpose

Do schizophrenics have altered glutamatergic function as evidenced by altered ketamine sensitivity?

Methods

Neuroleptic-treated schizophrenic subjects completed 2 test days during which they received ketamine (a bolus of 0.65 mg/kg/min for 1 minute followed constant infusion of 0.5 mg/kg/min for 1 hr.) or placebo in a double-blind, randomized and counterbalanced design. Measures of psychosis (Brief Psychiatric rating Scale: BPRS), perceptual alterations, frontal and temporal cortical, and motor function were administered.

Results

Ketamine increased 4-key positive symptom subscale scores (p = 0.07), 3-key negative symptom subscale scores (p = 0.06) and total scores (p = 0.004) of the BPRS. Ketamine also increased clinician-rated (p = 0.0001) and subject-rated (p = 0.0006) perceptual alteration scores. These results were compared to ketamine effects in healthy subjects who were pretreated with haloperidol 5 mg. There were differences in both the magnitude and quality of ketamine effects in neuroleptic-treated schizophrenics and neuroleptic-treated controls. For example, ketamine produced greater changes in scores of perceptual alterations, negative symptoms and positive symptoms in schizophrenics versus controls.

Conclusions

These data suggest altered ketamine sensitivity in schizophrenia and lend support to the hypothesis that altered glutamatergic function contributes to the neurobiology of schizophrenia. (Supported by the Department of Veterans Affairs.)

Toxicity of Mutant SOD-1 in Motor Neurons is Mediated Via Non-NMDA Ionotropic Receptors and Prevented by Calbindin D28K (GII-A)

Figlewicz, D.A.; Roy, J.; Minotti, S.; Dong, L.; and Durham, H.D.

Mutations in Cu/Zn-superoxide dismutase (SOD-1) are responsible for a familial form of amyotrophic lateral sclerosis (FALS). We have established a primary culture model which reproduces many features of this disease (J. Neuropathol. Exp. Neurol. 56: 523-530, 1997). Human SOD-1 cDNAs bearing mutations found in FALS patients (mSOD) were subcloned into pCEP4. mSOD constructs or pCEP4 "empty vector" were microinjected into nuclei of motor neurons in dissociated cultures of murine spinal cord, along with the marker dextran-FITC. A gradual loss of motor neurons injected with mSOD constructs occurred over 12 days [G93A (53 percent), G41A (54 percent), N139K (86 percent)]. Blockade of AMPA/kainate receptors by CNQX prevented death of motor neurons expressing mSOD. Since mutant protein was expressed only in motor neurons in this model, dysfunction of astrocytes or of neurons providing synaptic input could not have contributed to motor neuron death. Thus, normally non-toxic levels of glutamate receptor activation were sufficient to potentiate the toxicity of mSOD in motor neurons, particularly via non-NMDA ionotropic receptors.

The presence of Ca++-permeable AMPA receptors has been implicated in the preferential susceptibility of motor neurons in ALS, exacerbated by a deficiency of cytosolic Ca++ - binding proteins such as calbindin. In cultured motor neurons, dramatic neuroprotection was obtained by coinjecting calbindin-D28k and mSOD expression vectors: viability was indistinguishable from control cultures. Partial protection was also obtained by blockade of L-type voltage-gated calcium channels with nifedipine. Our results demonstrate the Ca++-dependence of mSOD-1 toxicity and implicate entry of Ca++ through both glutamate receptor channels and voltage-gated calcium channels in potentiating the toxicity of these SOD mutants in motor neurons.

Potential Involvement for the Plasma Membrane Calcium Atpases in Glutamate Induced Cell Death (GI-A)

Garcia, M.L.; Strehler, E.E.; Isackson P.J.; and Windebank, A.J.

Glutamate toxicity may be the final common pathway of several neuronal insults and neurodegenerative diseases. Delayed cell death, induced by excess glutamate, is mediated by calcium. As an initial assessment of the possible involvement of the plasma membrane calcium ATPases (PMCAs) in glutamate induced neurodegeneration, we have determined the effect of seizures upon PMCA mRNA expression. Kainic acid altered the expression within the hippocampal subfields of all PMCA mRNA isoforms, as determined by in situ hybridization. PMCA 1, 2 and 3 mRNAs exhibited hybridization below control levels 12 to 48 hours post-injection within CA1 and CA3 of the hippocampus. In the dentate gyrus, PMCA 2 mRNA hybridized below control levels 4 hours post-injection and exceeded control levels at 48 and 96 hours post-injection. The physiological significance of altering the expression of PMCA isoform 4 is now being investigated in a cell culture model. PC12 cells were transfected with an antisense (pCIneo-antiPMCA4) construct to decrease endogenous PMCA4 expression. In addition, PC12 cells were transfected with a full length sense (CMV-hPMCA4b-neo) construct to overexpress hPMCA4b. Clonal cell lines expressing these constructs have been established. Viability studies will determine the effects of altering PMCA expression on cell survival.

Changes in AMPA Receptor Subunit Expression in Ventral Motor Neurons After Spinal Cord Injury (GII-A)

Grossman, S.D.; Wolfe, B.B.; Yasuda, R.P.; and Wrathall, J.R.

Motor neurons are particularly susceptible to AMPA/KA mediated excitotoxicity. They undergo chronic neurodegeneration in amyotrophic lateral sclerosis and following spinal cord injury (SCI). Further, we found the AMPA/KA antagonist NBQX to be highly neuroprotective in a standardized model of SCI. We have now investigated changes in AMPA receptor subunits in ventral motor neurons following contusive SCI (10g x 2.5cm) at T8. Two groups of rats (n = 3 per group) were analyzed to determine changes in mRNA levels: Acute SCI at 24 hours postinjury (p.i.) and acute laminectomy controls. In situ hybridization was done using four [35S] end labeled antisense oligodeoxynucleotide probes specifically directed against GluR1, GluR2, GluR3, or GluR4. We analyzed grain counts from surviving motor neurons 4mm away from the epicenter of injury, where approximately 60 percent of ventral motor neurons are spared after SCI. We found a significant decrease in GluR2 mRNA (53 percent) at 24 hours p.i. compared to control levels. The decrease in GluR2 is specific since neither GluR3 nor GluR1 mRNA was significantly changed by 24 hours. These results are consistent with changes seen in protein levels, as determined by Western blot of thoracic tissue homogenates at 24 hours p.i., using antibodies against GluR1 and GluR2/3. Since the relative abundance of GluR2 is positively correlated to the calcium impermeability of the channel, acute downregulation could foster excitotoxicity. Alternatively, calcium influx through AMPA receptors may promote plasticity through synaptic strengthening, as is the case in long term potentiation. (Supported by NIH NS28130.)

Comparison of Subunit-Specific NMDA Receptor Channel Block by ADCI and Ketamine (GI-A)

Harty, P. and Rogawski, M.A.

ADCI ([(+/-)-5-aminocarbonyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5, 10-imine]), and ketamine (2-O-chlorophenyl-2-methylamino cyclohexanone), reduce NMDA-evoked currents by a channel blocking mechanism. Yet these two compounds exhibit very different behavioral and clinical profiles. Ketamine is a dissociative anesthetic similar to dizocilpine and phencyclidine, producing psychotomimetic effects at high doses. In contrast, ADCI is considered primarily an anti-convulsant, and exhibits little or no psychotomimetic effects. We hypothesized that these marked differences might be related to subunit-specific blocking properties of the two compounds. To test this idea, we used recombinant DNA and mammalian (HEK) cells to express NMDA receptors composed of subunits NR1a/NR2A, NR1a/NR2B or NR1a/NR2C. Using a rapid perfusion system, NMDA receptors of known subunit composition were exposed to NMDA with and without different concentrations of blockers. These data were used to construct and compare the sub unit-specific dose-response properties of the two compounds. In addition, the onsets and offsets of block produced by the drugs at the different heteromeric receptors were fit with exponential functions, and the time constants were incorporated into simple kinetic models of ligand binding.

Deafferentation Induced Cell Death in the Spinal Dorsal Horn is Potentiated by NMDA Receptor Stimulation But Not by GABAA or Glycine Receptor Inhibition (GII-B)

Jasmin, L. and Janni, G.

The neural pathways responsible for nociceptive transmission are not hard wired, but undergo structural and functional changes in response to neural and trophic signals. Events taking place in the periphery can trigger changes that can then modify sensory processing temporarily or permanently in the CNS. Deafferentation injury in particular leads to chronic intractable pain. We present evidence of cell death in nociceptive areas of the spinal cord after peripheral nerve section in adult rats, a phenomenon that could be a determinant in the irreversibility of symptoms and altered responses to therapy. Sciatic nerve section was associated with apoptotic and necrotic neuronal death in the lumbar superficial dorsal horn 24 hours after injury. NMDA (6.9 nmol) administered intrathecally at the time of sciatic n. section was followed by a 300 percent increase in the number of apoptotic cells. NMDA in sham operated rats, however, did not by itself cause any cell death.

Intrathecal administration of the GABAA antagonist bicuculline (40 to 80 nmol) or the glycine receptor antagonist strychnine (80 nmol) had no effect at doses that induce strong nociceptive behaviors. These results indicate that spinal nociceptive neurons acutely stimulated by peripheral nerve injury are more susceptible to NMDA excitotoxicity. It remains to be determined if the commitment to cell death occurs at the time of nerve injury as a result of massive depolarization, or if it occurs later as a result of either stimulus and/or deafferentation induced transcriptional and biochemical events, a phenomenon potentiated by NMDA receptor stimulation.

Activation of Hippocampal Glutamate Receptors in a Mouse Model of AIDS Dementia Complex (GII-A)

Keele, N.B.; Usherwood, P.N.R.; Espey, M.G.; Basile, A.S.; and Rogawski, M.A.

Whole-cell voltage clamp of cultured rat hippocampal neurons in vitro was used to study the effects of antibodies isolated from mice inoculated with the leukemia retrovirus LP-BM5 in vivo, a mouse model of AIDS (MAIDS). MAIDS mice share immunological and neurological effects observed in humans infected with HIV-1, including the neuropathology and cognitive impairments which manifest as AIDS dementia complex (ADC). Over-activation of glutamate receptors is implicated in ADC. This project tests the hypothesis that the MAIDS immune response yields antibodies that activate glutamate receptors in the brain, and characterize the mechanism of action.

The following observations were made:

1. MAIDS serum IgG and MAIDS brain IgG evoke inward current in hippocampal neurons, but not serum from control mice.
2. The MAIDS IgG-activated current (Imaids) is inhibited by CNQX, but more potently by the AMPA receptor-selective non-competitive antagonist GYKI 52466.
3. The reversal potential of Imaids is similar to that of kainate-activated current.
4. Imaids is sensitive to the AMPA receptor desensitization inhibitor, cyclothiazide (CTZ).
5. The concentration-response relationship of Imaids is an inverted-U shape, suggesting a CTZ-insensitive component of desensitization.
6. Imaids is not observed until hippocampal neurons are maintained 9 days in vitro (div) and is apparently maximal at 14 div, suggesting developmental regulation.
7. Membrane currents evoked by AMPA and MAIDS interact cooperatively.
8. These data are consistent with retrovirus-induced antibodies that activate AMPA receptors in the brain. The antibody GluR activation, together with potential cooperativity with endogenous ligands, may participate in the excessive glutamatergic tone underlying the neurological deficits of ADC.

Metabotropic Glutamate Receptors, Midbrain Dopamine and the Generation of Locomotor Activity (GI-B)

Kim, J. and Vezina, P.

Interactions between excitatory amino acid (EAA) and dopamine (DA) pathways in the basal ganglia have been known for some time to contribute importantly to the generation of motor behaviors. In particular, the role played by ionotropic glutamate receptors (iGluRs) in such interactions and in the production of locomotion has received considerable attention particularly in brain areas such as the ventral tegmental area (VTA) where EAA afferants are known to modulate the activity of DA neurons and the nucleus accumbens (NAcc) where descending EAA projections and ascending DA mesencephalic projections come in close apposition to each other and co-innervate intrinsic neurons projecting to motor output regions. Recently, the growing importance of the metabotropic glutamate receptor (mGluR) in the generation of motor behaviors and various forms of plasticity has begun to emerge. The known coupling of the mGluR to second messenger systems and its demonstrated role in the long-term modulation of synaptic transmission make it a logical candidate not only for the generation of locomotion involving EAA-DA interactions but also for the induction and expression of locomotor plasticity involving these neurotransmitters. Evidence is presented supporting a role for mGluRs in the generation of DA-dependent locomotion as well as in one form of locomotor plasticity: the sensitization of locomotor activity by psychomotor stimulant drugs.

Normal Sensitivity to Spermine of NMDA-Evoked Responses from Trisomy 16 Mouse Embryonic Cultured Hippocampal Neurons (GII-A)

Klein, R.C.; Siarey, R.J.; Rapoport, S.I.; Castellino, F.J.; and Galdzicki, Z.

It has been proposed that polyamines (e.g. spermidine, spermine) play an important role in brain development, function, and in certain neurodegenerative conditions (8). Down syndrome (DS) is the most common, human autosomal abnormality characterized by mental retardation, hypotonia, and early onset of Alzheimer's disease (AD). McCoy et al. (1982) (3) reported a decreased content of polyamine in DS lymphocytes stimulated by Concanavalin A. In addition, lower levels of spermidine and spermine were found in frontal cortex of DS and AD patients (5).

The trisomy 16 (Ts16) mouse is considered a genetic model of DS, as there is a substantial homology between the long arm of human chromosome 21 and the distal portion of mouse chromosome 16. No AD-type phenotypic changes were found in Ts16 grafts (6), however functional changes, similar to AD abnormalities, have been reported (2, 4). In addition, changes in densities of voltage-dependent sodium and calcium currents in Ts16 hippocampal cultures have been reported (1, 7).

The NMDA receptor constitutes a second major route of calcium entry into hippocampal neurons, therefore we investigated NMDA-evoked currents and the sensitivity to spermine. In both Ts16 and diploid neurons, NMDA (6-150 M) evoked similar inward currents. All principle properties, such as the reversal potential, the minimum current, and the dose response of NMDA-evoked currents showed no significant differences between Ts16 and diploid neurons. The current evoked by NMDA was potentiated in a dose dependent manner for all doses of spermine (< 1 mM) and this potentiation was not voltage-dependent. The dose-responses of spermine were similar for both Ts16 and diploid neurons. These results further support our previous data that NMDA-evoked currents are not altered in Ts16 neurons.

References

1. Galdzicki, Z.; Coan, E.J.; Rapoport, S.I.; and Stoll, J. Mol Brain Res, in press, 1998.
2. Holtzman, D.M.; Li, Y.W.; DeArmond, S.J.; McKinley, M.P.; Gage, F.H.; Epstein, C.J.; and Mobley, W.C. Proc Natl Acad Sci USA 89:1383-1387, 1992.
3. McCoy, E.E.; Strynadka, K.; Pabst, H.F.; and Crawford, J. Pediatr Res 16:314-317, 1982. 
4. Nelson, P.G.; Fitzgerald, S.; Rapoport, S.I.; Neale, E.A.; Galdzicki, Z.; Dunlap, V.; Bowers, L.; and Agoston, D. Proc Natl Acad Sci USA 94:12644-12648, 1997. 
5. Seidl, R.; Beninati, S.; Cairns, N.; Singewald, N.; Risser, D.; Bavan, H.; Nemethova, M.; and Lubec, G. Neurosci Lett 206:193-195, 1996. 
6. Stoll, J.; Balbo, A.; Ault, B.; Rapoport, S.I.; and Fine, A. Brain Res 610:295-304, 1993. 
7. Stoll, J., and Galdzicki, Z. Int J Dev Neurosci 14:749-760, 1996. 
8. Williams, K. Cell Signal 9:1-13, 1997.

Altered Responses to the NMDA Antagonist, Ketamine, in Recently Detoxified Early Onset Alcoholics (GI-B)

Krystal, J.H.; Petrakis, I.L.; D'Souza, D.C.; Trevisan, L.; Karper, L.; Cooney, N.; and Charney, D.S.

At doses commonly associated with intoxication, antagonism of NMDA glutamate receptors is among the most prominent actions of ethanol. The purpose of this study is to begin to study the clinical implications of the NMDA antagonist actions of ethanol: 1) do alcoholic patients recognize NMDA antagonists as ethanol-like? and 2) are there changes in the sensitivity to ketamine that might be consistent with post-mortem data describing increases in NMDA receptor binding in these patients?

Methods

Recently detoxified male alcoholic patients (n = 20) and male controls (n = 15) completed 3 test days involving the i.v. administration of placebo, ketamine 0.1 mg/kg, or ketamine 0.5 mg/kg over 40 min. under double-blind conditions.

Results

Ketamine effects were rated by patients as more similar to ethanol than to cocaine or marijuana. The 0.1 mg/kg was rated as similarly in intensity to approximately 1 standard ethanol drink and the 0.5 mg/kg dose was rated as similar in intensity to approximately 9 standard ethanol drinks. The psychotigenic, perceptual, cognitive, and neuroendocrine effects of ketamine were significantly blunted in patients relative to controls. However, the euphoric effects of ketamine were not reduced in patients.

Implications

These data suggest the NMDA antagonist properties of ethanol figure prominently in the behavioral effects of this drug. Reduced ketamine sensitivity in recently detoxified alcoholics may be consistent with cross-tolerance between ethanol and ketamine. Lastly, the reward valence of ketamine appears to be altered in recently detoxified alcoholics: they experience markedly reduced levels of unpleasant ketamine effects (psychotigenic, perceptual, cognitive, anxiogenic) relative to controls, but preserved euphoric effects. This relative enhancement in the overall rewarding properties of ethanol may contribute to heavy drinking, and thus relapse, in recently detoxified patients. (Supported by NIAAA and the Department of Veterans Affairs.)

Disruptive Effects of the NMDA Antagonist, Ketamine, Upon Sensorimotor Gating in Humans (GII-B)

Krystal, J.H.; Karper, L.; Abi-Saab, D.; Belger, A.; D'Souza, D.C.; Charney, D.S.; and Grillon, C.

Previous clinical research conducted by our group and others suggests that schizophrenia is associated with impairments in sensorimotor gating, i.e., deficits in the "pre-pulse" inhibition (PPI) of the startle response that are associated with impairments in focusing attention, i.e., filtering environmental stimuli. Previous preclinical research has suggested that NMDA receptor deficits contribute to PPI deficits and that these deficits are preferentially reduced by atypical rather than typical neuroleptics. In addition, characterization of ketamine effects on PPI may help to characterize the cognitive effects of drugs, such as ethanol, that block NMDA receptors.

Methods

Healthy subjects (n = 20) completed 2 test days, involving i.v. infusion of placebo or ketamine (bolus 0.23 mg/kg followed by 0.5 mg/kg/hr). The acoustic startle response was measured (San Diego Instr. Startle-SR) and other assessments were completed. Each startle trial consisted of 6 blocks (collapsed to 3 for analysis) containing 6 stimuli involving all combinations of 3 pre-pulse intensities (no prepulse, 48 dB and 51 dB) and 2 stimulus intensities (100 dB and 113 dB).

Results

Ketamine reduced startle amplitude, but did not significantly effect habituation. The PPI analysis focused on the first of the three blocks of stimuli. Ketamine significantly reduced pre-pulse inhibition of the startle response (drug by prepulse by stimulus intensity interaction by order of test day: F[2,32] = 4.2, p = 0.02). These effects were greatest for the weaker pre-pulse and pulse stimulus intensities where the magnitude of PPI was reduced by approximately one-half (mean placebo: 91 units vs. mean ketamine: 47 units). Cognitive data are currently under analysis.

Implications

These data suggest ketamine depresses the acoustic startle response in humans. In addition, ketamine reduces PPI of the startle response in a fashion that is dependent on several factors. Previous data from our group described increases in distractibility associated with ketamine administration in humans. These data are consistent with the view that deficits in NMDA receptor function that reduce sensory gating may contribute to impairments in the control of attention. (Supported by NARSAD and the Department of Veterans Affairs.)

GluR5 Kainate Receptor Mediated Synaptic Transmission in Rat Basolateral Amygdala in Vitro (GI-A)

Li, H.; Post, R.M.; and Rogawski, M.A.

This study examines a non-NMDA and non-AMPA receptor mediated excitatory postsynaptic response evoked in the basolateral amygdala (BLA) by the stimulation of the external capsule (EC). The 2,3-benzodiazepine AMPA receptor-selective allosteric antagonists GYKI 52466 and GYKI 53655 (50 &M) depressed single shock EC-BLA synaptic responses by 66 Å 3 percent and 83 Å 3 percent respectively but fully blocked synaptic responses evoked by stimulation of basal amygdala (BA). Applications of train stimulation to the EC in the presence of GYKI 52466-containing cocktail and GYKI 53655-containing cocktail evokes a further increase in the amplitude of synaptic response range from 8.4 Å 1.4mV and 7.1 Å 1.1mV respectively. Thus in the presence of GYKI 52466-containing cocktail this train evoked synaptic responses are largely blocked by 20 &M CNQX at 85.3 Å 3.5 percent. Meanwhile, in the presence of GYKI 53655-containing cocktail train evoked synaptic responses are fully blocked by 10 &M LY293558 at 94.5 Å 2.3 percent suggesting this kainate receptor mediated synaptic transmission is mediated predominantly by GluR5 receptor activation in the amygdala.

NMDA Receptor Subunits Distribution in Cultured Cortical Neuron (GI-A)

Li, J. and Vincini, S.

Using antibodies against distinct NMDA receptor (NR) subunits, we report that in developing rat cortical neurons in culture NR1 and NR2B subunits display high levels of expression within the first week while the NR2A subunit is barely detectable at 7 days in vitro (DIV) and gradually increased to mature levels. Immunocytochemical analysis indicated that clusters of NR1 and NR2B subunits were observed as early as DIV3, while NR2A clusters were rarely observed before DIV10. In some neurons NR subunit clusters failed to develop, and only diffuse microclusters were observed. At DIV18 NR2A and NR2B clusters well colocalize with those of NR1 subunits however the extent of colocalization between NR2A and NR2B subunits was cell specific as the antagonism of NR-mediated synaptic currents by subtype selective drugs. Synapse formation as indicated by synaptophysin staining and electrophysiological recordings, was observed as early as 72 hours after plating. However, in several neurons at ages less than DIV5 where synapses were scarce, NR2B and NR1 clusters were abundant. We also investigated the distribution of NR subunit clusters in relationship to distinct neuronal compartment such as axon, dendritic shafts and dendritic spines by transient transfection of green flourescent protein (GFP) into cultured cortical neurons. We provide a direct evidence that the NR2B subunit clusterize on both dendritic shafts and spines. At the same time, we also observed that NR2B subunit is poorly colocalize with GluR1 subunit. These data provide a direct evidence of cell and compartment dependent expression and distribution of specific NR subunits.

Accumulation of Glutamate in Experimental Diabetic Retinopathy (GI-A)

Lieth, E.; Ratz, M.J.; Tanase, D.; Antonetti, D.A.; Gardner, T.W.; and Barber, A.J.

The early pathophysiology of diabetic retinopathy and the involvement of neural and vascular malfunction are poorly understood. We recently showed that diabetes impairs glial glutamate metabolism in retinas of rats soon after the onset of hyperglycemia. Elevated retinal glutamate can cause excitotoxic damage to ganglion cells of the retina. Here we investigate the possibility that glutamate accumulates in retinas in short-term diabetes. Streptozotocin diabetic rats and age-matched normal rats were housed for 1, 2 or 3 months. Retinas were dissected and homogenized to measure glutamate by luminometry. Also, glial fibrillary acidic protein (GFAP) was measured by ELISA to assess glial reactivity. After 1 month of diabetes no significant increase could be detected either in GFAP immunoreactivity or in retinal glutamate. After 3 months GFAP maximally increases by 5-fold (p < 0.04) and glutamate rises 1.6-fold (p < 0.04). These findings demonstrate for the first time that retinal glutamate rises in short-term diabetes. Elevated retinal glutamate could therefore damage neurons throughout the course of diabetes, eventually leading to vision loss.

Glutamatergic Modulation of kappaB-Binding Transcription Factors: The NF-kappaB Red Herring (GI-B)

Mao, X.; Moerman, A.M.; and Barger, S.W.

Studies of kappaB-binding factors in neural tissues have concluded that the transcription factor NF-kappaB is constitutively active in neurons or that its activity is elevated by glutamate. In contrast, we find no evidence of bona fide NF-kappaB DNA-binding activity in neurons under basal or glutamate-stimulated conditions. In highly enriched neuron cultures from hippocampus and neocortex, we detect a constitutive kappaB-binding activity that is distinct from known members of the NF-kappaB family by antigenicity, electrophoretic mobility, physiological regulation, and biochemical behavior. We have tentively named this factor NKBF. The activity of NKBF was rapidly diminished by toxic exposures of glutamate. Activation of both NMDA and non-NMDA ionotropic receptors was required for the full attenuation of NKBF activity. This effect also involved calcium influx and was sensitive to cyclosporin A. Bona fide NF-kappaB activity was induced by glutamate in cocultures containing large numbers of glia but not in glia cultured alone. When kappaB binding activity was assayed separately in the neurons or glia from a coculture, glia accounted for all of the bona fide NF-kappaB activity. These data indicate that the kappaB-binding transcription factor in neurons is negatively regulated by glutamate, in a manner correlated with but preceding neurotoxicity. This conclusion strengthens our previous data indicating that neuronal kappaB-dependent transcription is correlated with neuronal survival in general and excitoprotection in particular. (Sponsored by funds from NINDS (NS35872), the Alzheimer's Association, and the Inglewood Foundation.)

Novel NMDA/Glycine Site Antagonists Prevent Cocaine-Induced Toxicity and Locomotor Sensitization (GI-B)

Matsumoto, R.R.; Brackett, R.L.; Brown, M.; Pouw, B.; Blyden, J.F.; and Kanthasamy, A.G.

No effective treatments exist for cocaine overdose and addiction. Since excitatory amino acid mechanisms may be involved in cocaine toxicity and abuse, novel NMDA/glycine site antagonists (ACEA-1021, ACEA-1031, ACEA-1328, each an "ACEA compound") were tested for their ability to attenuate cocaine-induced behavioral toxicity and locomotor sensitization in male, Swiss Webster mice. Pretreatment with an ACEA compound (0-60 mg/kg, i.p.) prior to a convulsive (60 mg/kg, i.p.) or lethal (125 mg/kg, i.p.) dose of cocaine significantly attenuated cocaine-induced convulsions and lethality. Protection was also produced by other NMDA/glycine site antagonists (7-chlorokynurenic acid, R(+)-HA-966) and structural analogs (DCQX). Structurally-related quinoxalinediones that lack interactions with NMDA/glycine sites (5-nitroquinoxalinedione, NBQX) failed to prevent cocaine-induced toxicity. The NMDA/glycine site agonist D-cycloserine totally prevented the protective effects of the ACEA compounds, confirming an action through NMDA/glycine sites. Significantly, i.p. administration of an ACEA compound after a normally lethal dose of cocaine prevented death in a significant number of animals (50 to 86 percent protection). Deaths were prevented by ACEA-1021 even when it was administered as a post-treatment in mice first exposed subchronically to cocaine for 7 days before receiving a normally fatal cocaine overdose. At doses that themselves did not alter spontaneous locomotion, concomitant administration of the ACEA compounds further prevented the development of locomotor sensitization. Subchronic pre-exposure to an ACEA compound for 5 days also prevented the development of locomotor sensitization, indicating prophylactic potential. Similarly to ACEA-1021 in human clinical trials (see related abstract), the compounds were well-tolerated, and may have the potential to treat cocaine abuse and overdose.

In Vivo Effects of Biperiden and Diazepam on Brain Cholinergic and Aminergic Neurotransmitters During Soman-Induced Seizures (GI-A)

Morris, J.L.; McDonough, J.H.; Sozio, S.M.; and Shih, T-M.

Guinea pigs, surgically implanted with cortical EEG electrodes and caudate nuclear-microdialysis probes, were administered pyridostigmine 30 minutes before the organophosphorus cholinesterase inhibitor soman [GD] and atropine/2-PAM. Five minutes after seizure onset, an antiseizure ED50 for the GABA agonist diazepam [DZ] or the antimuscarinic biperiden [BP] was given, and animals were separated into seizure not terminated [ON] and seizure terminated [OFF] groups. Soman-increased acetylcholine [ACh] was decreased by DZ and BP after 30 minutes. While ACh levels in DZ ON and OFF animals did not differ, ACh levels in BP OFF animals were decreased from GD alone and BP ON animals. Soman decreased glutamate [Glu] to 60 percent of control for the first 75 minutes after which Glu returned to control. Both DZ and BP decreased Glu in ON and in OFF animals, especially between 2-4 hours of soman exposure, but Glu levels were similar in ON and OFF animals with both drugs. Soman decreased GABA to 70 percent of control with no changes from BP or DZ. These results suggest that BP and DZ may affect caudate nuclear soman-induced seizure activity by other mechanisms in addition to muscarinic blockade and GABA agonism, respectively. (This work was performed while Dr. Morris held a National Research Council-USAMRICD Research Associateship.)

Long-Term Potentiation in Vivo Increases Rat Hippocampal Tenascin-C Expression (GI-A)

Nakic, M.; Manahan-Vaughan, D.; Reymann, K.G.; and Schachner, M.

We investigated the expression of the extracellular matrix glycoprotein tenascin-C after induction of long-term potentiation (LTP) by high frequency tetanization (HFT) in the rat dentate gyrus in vivo. Expression of tenascin-C was evaluated at the mRNA and protein levels by in situ hybridization and immunocytochemistry, respectively. Whereas no tenascin-C mRNA was detectable in control animals, an increase in tenascin-C mRNA levels was observed in the granule cell layer of the dentate gyrus 4 hours after HFT. Twenty-four hours after HFT, tenascin-C mRNA returned to control levels. Expression of tenascin-C protein 4 hours after HFT followed that of controls in that tenascin was detectable in the strata oriens and radiatum of CA1, in the molecular layer and within a narrow area at the inner surface of the granule cell layer in the dentate gyrus. However, 24 hours after HFT, additional patches of tenascin-C immunoreactivity were observed in the molecular layer of the dentate gyrus. No increase in tenascin mRNA or protein levels were detected in control animals that received no stimulation, low-frequency stimulation or HFT in the presence of the N-methyl-D-aspartate receptor antagonist D(-)-2-amino-5-phosphonopentanoic acid (AP5) or the metabotropic glutamate receptor antagonist (R,S)-a-methyl-4-carboxyphenylglycine (MCPG). These observations implicate a role for tenascin-C in N-methyl-D-aspartate and metabotropic glutamate receptor dependent changes accompanying induction and/or maintenance of LTP.

Key words: extracellular matrix, recognition molecule, synaptic plasticity, mGluRs, NMDA, long-term potentiation.

Identification Of NMDA Receptor Dominant Mutations and Study of Apoptotic Function (GI-A)

Rameau, G. and Ziff, E.

Excitotoxic death of neurons occurs following over stimulation of NMDA-type glutamate receptors (NMDAR). I have shown that transient expression of the NMDAR leads to cell death in chinese hamster ovary (CHO) cells. MK-801, a specific NMDAR antagonist, can block this effect. I have quantitated cell killing by measuring the activity of a co-transfected firefly luciferase expression plasmid. To examine the function of the NMDAR, the NR1 subunit was mutated by PCR mutagenesis. Asparagine at amino acid position 616 and serine at position 617 in the receptor pore were substituted for arginine (N616R) and glutamine (S617Q) respectively. These mutations are known from previous work to decrease fluxes of calcium ions. A survival curve obtained by independently expressing the wild type and mutants receptors showed decrease mutant receptor excitotoxicity in comparison to the wild type. Coexpression of mutants with wild type subunits also exerted dominant negative effects and reduced the excitotoxicity of the wild type receptors.

I have also employed this system to examine the role of apoptosis in excitotoxic cell death induced by the NMDAR. I have shown that cells expressing the NMDAR stained positive for annexin V and displayed nuclear DNA-laddering fragmentation that correlate with apoptotic cell death. Similarly, cortical, hyppothalamus and brain stem derived neurons bind Annexin V and produced the same characteristic DNA laddering when exposed to NMDA. In addition, the ICE-protease inhibitors including the baculovirus derived p35 and the pox-virus CramA proteins, block NMDA receptor-dependent cell death in a dose responsive manner, demonstrating that death occurs by an apoptotic mechanism.

I have studied roles of Ca2+-dependent processes and receptor binding proteins in excitotoxicity. Preliminary data have shown that expression of a constitutively activated form of calcineurin increases NMDAR dependent cell death, and this is potentiated further by coexpression of PSD95. These results suggest involvement of calcineurin in the NMDAR-dependent cell death signal mechanism. (Supported by an Aaron Diamond Foundation Postdoctoral Fellowship.)

The Effect of EAA Modulators on Acute Naloxone Precipitated Opiate Withdrawal (GI-B)

Rosen, M.I. and Kosten, T.R.

NMDA receptor antagonists have been reported to decrease the development of tolerance and physical dependence in opiate-treated animals; given acutely these agents as well as an AMPA antagonist attenuate opiate withdrawal. However, agents acting at the PCP-site of the NMDA receptor (e.g., MK801, ketamine) show many common features in animal behavioral paradigms, and are likely to have some PCP-like side effects. An alternative approach has been to develop agents that attenuate excitatory amino acid (EAA) activity but do not act directly at the PCP recognition site. Our group has developed an efficient within-subjects methodology to study the effects of EAA modulators on acute opiate withdrawal. Serial naloxone challenge tests in the same opioid dependent subject yield reliable measures of opiate withdrawal. As would be expected for a paradigm sensitive enough to detect an effective anti-withdrawal agent, the naloxone challenge paradigm detected significant attenuation of specific withdrawal signs by clonidine pre-treatment in 7 subjects. Negative controls have not been systematically tested, but gamma-hydroxybutyric acid had minimal effects on precipitated withdrawal in this paradigm. Results will be presented from putative excitatory amino antagonists tested to date in this paradigm including cycloserine (a putative partial agonist at the strychnine-insensitive glycine site of the NMDA receptor), lamotrigine (a marketed anticonvulsant that attenuates glutamate release), and dextromethorphan (a competitive antagonist at the PCP site of the NMDA receptor).

The Activation of Metabotropic Glutamate Receptors Protects Nerve Cells from Oxidative Stress (GI-A)

Sagara, Y. and Schubert, D.

Metabotropic glutamate receptors (mGluRs) have been implicated in a variety of cellular responses to glutamic acid, including the modulation of long term potentiation, glial activation, and excitotoxicity. The work described in this poster extends the role of mGluRs to include protection from oxidative stress induced programmed cell death. The activation of group I mGluRs protects nerve cells via the inositol-1,4,5 triphosphate (IP3) pathway from cell death initiated by oxidative glutamate toxicity. mGluR1 and 5 antagonists potentiate glutamate toxicity, while selective group I agonists are protective. These glutamate analogues regulate IP3 mass accumulation in accordance with their ability to protect cells, and protection appears to take place at the level of glutathione metabolism. Short term exposure of cells to low concentrations of glutamate desensitizes cells to a subsequent challenge from glutamate. Glutamate exposure also upregulates the expression of mGluR5 in HT22 cells and mGluR1 in cortical primary cultures. Group I mGluR agonists also protect cells from death programs initiated by glucose starvation and cystine deprivation. These data show that the activation of group I mGluRs elicits a protective response in neurons which is able to block at least some forms of cell death.

Developmental Phencyclidine (PCP) Exposure: Sensorimotor Effects (GII-A)

Scalzo, F.M.

Our laboratory has been investigating the residual motor effects of developmental phencyclidine (PCP) exposure and has identified critical periods of early postnatal development for PCP-induced alterations in the locomotor response to both PCP and MK-801. In an attempt to determine if developmental exposure to PCP results in alterations in sensorimotor function, we measured the reflex modulation of the acoustic startle response in rats following PCP exposure during two periods of development. Rats were treated with saline or 7.5 mg/kg PCP on postnatal days (PNDs) 4-17 or 24-37. Startle responding was measured 9 and 19 days after PCP treatment. Treatment with PCP on PNDs 4-17 resulted in a shorter response latency and decreased prepulse-inhibition (PPI) using, 40, 60 and 80 msec stimulus intervals. Treatment with PCP on PNDs 24-37 did not affect PPI. The results suggest that subchronic neonatal PCP exposure results in alterations in neural systems mediating the startle response and the modulation of the response. (Supported by NIDA Grants DA-06319 & DA-08240.)

Changes in NMDAR1 and Glur2 Receptor Subunit Populations With Sensorineural Hearing Loss in The C57Bl/6 Mouse Model of Presbycusis (GI-A)

Erulkar, J.S. and Schwartz, I.R.

This study sought to identify changes in two ionotropic glutamate receptor subunits in brainstem auditory nuclei in 8wk (normal hearing) and 12m (severely hearing impaired) C57Bl/6 mice, a well documented model of presbycusis (Mikaelian 1979, Willott 1986). We examined the distributions of two subunits: NMDAR1 (an obligate member of functional NMDA receptors) and GluR2 (which confers Ca+ impermeability to the pentameric AMPA receptor). Hearing levels were confirmed with auditory brainstem responses. Animals were perfused with a 4 percent paraformaldehyde, 0.1 percent glutaraldehyde fixative. Cryostat sections of brainstem were incubated with antibodies to the NMDAR1 subunit (PharMingen) and the GluR2 subunit (gift of Dr. Wenthold), processed with the Vector Elite ABC procedure. There were significant differences in the NMDAR1 and GluR2 staining patterns of both neurons and neuropil of layers in the dorsal cochlear nucleus (DCN). The 8wk mice displayed intense NMDAR1 staining of fusiform cells and cells of the deep layer. In contrast, cells in 12m C57s were lightly stained throughout the DCN. The intensity of staining of the molecular layer neuropil also declined. There was little change in the NMDAR1 staining pattern of the octopus cell area (OCA) of the posterior ventral CN. Both groups showed similar staining in the somata and dendritic trees of octopus cells. Two regions receiving primary afferent input (DCN deep layer and OCA) showed different changes in their staining patterns. There was a decrease in the density of GluR2 immunoreactive cells, in the staining intensity of the cell bodies and dendrites of the fusiform layer and in the staining intensity of the neuropil of the molecular layer between 8wk and 12m. Changes in the NMDAR1 and GluR2 staining across the CN did not show a relationship to the underlying tonotopic organization, as might be expected if CN changes were secondary to progressive cochlear degeneration from base to apex. Our data suggest that genetically controlled changes in NMDA and GluR2 receptors may provide an explanation for the observation that deficits in the processing of auditory information in presbycusis is greater than predicted by the peripheral loss (Frisina & Frisina, 1997). (Supported by NIH grant DC00132.)

Pre-Synaptic NMDA Receptors in Gabaergic Terminals: Ultrastructural Evidence and Species Difference Between Rats and Monkeys (GI-A)

Paquet, M. and Smith, Y.

On the basis of current anatomical and electrophysiological data, NMDA receptors (NMDAR) are commonly seen as post-synaptic receptors. Although some electrophysiological and pharmacological evidence suggest that NMDAR may also have pre-synaptic effects in certain brain regions, immunocytochemical findings revealed that NMDAR subunits immunoreactivity is largely confined to post-synaptic structures. The findings of the present study provide anatomical evidence for the existence of pre-synaptic NMDAR1 subunit immunoreactivity in large subpopulations of axon terminals in the paraventricular (PVH) and arcuate (ARH) nuclei of the hypothalamus, the paraventricular nucleus of the thalamus (PVT) and the bed nucleus of the stria terminalis (BST) in rats. A smaller density of NMDAR1-positive terminals were also found in the basal forebrain, the central nucleus of the amygdala, the rostral hypothalamic region, the ventral tegmental area and the periaqueductal gray.

At the electron microscopic level, NMDAR1-immunoreactive terminals in the PVH, ARH and BST had the ultrastructural features of inhibitory terminals and, indeed, displayed GABA immunoreactivity. It is worth noting that: (1) the NMDAR1 terminal staining was not found in monkeys, and (2) it only showed up with the polyclonal antiserum raised against the C-terminus of the rat NMDAR1 subunit (Petralia et al., J. Neurosci. 14: 667). Two monoclonal antibodies raised against different sites of the NMDAR1 peptide (Siegel et al., PNAS 91: 564; Nash et al., J. Neurochem. 69:485) did not lead to this terminal labelling.

In conclusion, our findings strongly suggest that NMDAR may act as pre-synaptic heteroreceptors in subpopulations of GABAergic terminals in the central nervous system. (Supported by NIH Grant RR-00165 and Dept of Neurology, Emory University.)

MK-801 Causes Terminal Degeneration in Widespread Areas of Rat Brain (GII-A)

Sparenborg, S.; Switzer III, R.C.; Forster, J.; and Filbert, M.G.

Single sc injections of the NMDA antagonist MK-801 (0.1, 0.3, or 0.5 mg/kg) were given to female rats 6, 14, 24, or 96 hours before sacrifice. Neuronal degeneration was evaluated in one brain hemisphere with the cupric-silver method. A single 1-micron-thick methacrylate section from the other hemisphere, containing the retrosplenial cortex, was stained with toluidine blue for visualization of neuronal vacuoles. Rats treated with the two higher doses of MK-801 and sacrificed 6 hours later had vacuoles in the cytoplasm of retrosplenial neurons. The mean number of neurons with vacuoles per section was 0, 0, 10, and 30, respectively, in the control, low, mid and high dose groups. Vacuoles were not found in rats with longer survival times. Although only the higher doses of MK-801 caused vacuolation, all doses caused synaptic terminal degeneration (TD) in many brain areas. At only 6 hours after drug administration, TD appeared in the superficial (I-IV) layers of retrosplenial cortex, in occipital and parietal areas processing vision and audition, and in frontal (motor) association areas. In the hippocampus, TD was mild to severe in the molecular layer of the dentate gyrus and in stratum lacunosum moleculare of CA1. These effects generally increased in severity with longer survival times and were mildly dose-related. Dead neurons were noted in the piriform cortex of some rats given the low dose of MK-801 and sacrificed 14 or 24 hours later. However, silver staining was virtually absent from all brain areas of low-dose rats sacrificed after 96 hours indicating that degenerated terminals and soma had been cleared from the brain. Many dead cell bodies were visible in retrosplenial cortex of mid- and high-dose rats sacrificed after 24 and 96 hours and were occasionally found in piriform and entorhinal cortices. TD represents the permanent loss of inter-neuronal communication through the affected synapses. Widespread TD and the loss of cells in the piriform cortex indicate that MK-801 produced a long-lasting toxic effect at a dose lower than that which produced vacuoles. (Supported by USAMRICD.)

Kainate Antagonists and Chronic Pain (GII-B)

Gu, Z.Q.; Kajander, K.; Maccecchini, M.; and Sturgess, M.A.

Previous efforts at Bearsden Bio (formerly Symphony Pharmaceuticals) had identified (2S, 4R)-4-methylglutamate (SYM 2081) as a novel and potentially promising kainate receptor antagonist. Recent work in our laboratories has developed our understanding of the interaction of this compound with kainate receptors. Data will be presented that show that kainate receptors are a promising therapeutic target for the treatment of chronic pain, and that SYM 2081 is a novel and effective agent for attenuating sensitivity in established animal models of acute and chronic pain.

Cellular Neuroanatomy of NMDA NR1 Splice Variants (GI-A)

Tanaka, M.; Caudle, R.M.; Benoliel, R.; Finegold, A.A.; and Iadarola, M.J.

The primary transcript of the NMDA NR1 subunit undergoes alternative splicing at exons 5,21,22, also called N1 cassette, C1 cassette, C2 cassette respectively, leading to different sets of subunits with different physiological properties. In order to study the distributions and the functions in vivo, we generated polyclonal antibodies affinity purified against exons 5,21,22, and the alternative C-termini when the exon 22 is spliced out. Immunocytochemical staining with these antibodies against the splice variants showed distinct localization. The immunoreactivity of N1 cassette were restricted to the perikarya and the proximal dendritic shaft both in spinal cord and brain. N1 positive cells were located throughout spinal cord and motorneurons were stained strongly. Subunits with the alternative C-termini (C2-) showed a similar subcellular distribution: the cell bodies and proximal dendrites.However , in spinal cord the C2- positive cells clustered around central canal and the motorneurons did not show immunoreactivity. In contrast, C2 containing subunits exhibited a wide spread localization throughout positive cells. This was especially evident in the purkinje cells in cerebellum where immunoreactivity was observed in the perikarya to the ends of the dendritic arbor.Subunits containing the C1 cassette were nearly exclusively expressed in the dendrites of the brain and very low to absent in spinal cord. These data suggest a selective recruitment of NR1 subunits to specific and generalized subcellular distributions in neurons that may be determined by alternative splicing not only of exon 21 but also of exons 5 and 22.

Glutamate Cascade from Metabotropic Glutamate Receptor to Immediate Early Gene Expression in Rat Striatal Neurons: Implication for Psychostimulant Dependence (GI-B)

Mao, L. and Wang, J.Q.

Metabotropic glutamate receptors (mGluR) are coupled to multiple intracellular second messenger systems through G-proteins and expressed by medium spiny projection neurons in the rat striatum. Unlike ionotropic glutamate receptors which mediate rapid synaptic transmission, mGluRs are more important for relatively long-lasting modulation of neuronal metabotropic activities, including gene expression. In chronically-cannulated rats, microinjection of a selective mGluR agonist, ACPD, into the rat striatum elevated immediate early gene c-fos and zif/268 mRNA expression in the injected dorsal striatum as revealed by quantitative in situ hybridization. The elevation of either mRNA was dose-dependent and the induction of c-fos at each dose surveyed was more prominent than that of zif/268. No behavioral alteration was observed after ACPD injection at any dose. Two mRNA induction was rapid and transient, as their increases became evident as early as 30 min, reached a peak at 1 h, and returned to normal levels 3 (c-fos) or 6 (zif/268) h, after ACPD injection. Selective mGluR antagonist, MCPG, was able to attenuate ACPD-stimulated c-fos, but not zif/268, expression. Pretreatment with the ionotropic NMDA receptor antagonist, CPP, had no effect on ACPD-stimulated c-fos expression, but partially attenuated ACPD-stimulated zif/268 expression. D1 dopamine receptor antagonist, SCH23390, did not alter the ability of ACPD to induce the 2-gene expression. The data demonstrate a difference between striatal c-fos and zif/268 gene expression in response to specific mGluR activation. The c-fos elevation was independent of D1 dopaminergic and NMDA glutamatergic transmission whereas zif/268 induction was mediated, at least in part, by NMDA receptors. These results suggest a prominent involvement of mGluRs in cellular gene expression which is thought to be an essential component for neuroplasticity underlying long-lasting action of psychostimulant exposure. (Supported by NIDA DA10355 and a Young Investigator Award from NC Governor's Institute and a research grant (4150486810) from Latham Trust of Crestar Bank.)

Metabotropic Glutamate Receptors as Novel Targets of Therapeutic Agents for Substance Addiction (GI-B)

Wang, J.Q.

Metabotropic glutamate receptors (mGluR) are densely expressed by striatonigral and striatopallidal projection neurons in the striatum. Activation of mGluRs in this brain region alters local transmitter release and behaviors of experimental animals. Of particular interest is that mGluRs regulate transcription factor and neuropeptide gene expression in the striatal neurons through their connections with multiple intracellular effectors. This prominent involvement of mGluRs in overall cellular activity is significant for development of neuroplasticity underlying long-term adaptive changes in cellular physiology related to a variety of neurological disorders. In our recent studies concerning role(s) of mGluRs in mediation of psychostimulant amphetamine actions, we found that bilateral icv injection of mGluR antagonist, MCPG, markedly attenuated acute amphetamine-stimulated transcription factors, c-fos and zif/268, mRNA expression in the rat striatum, with sparing amphetamine- stimulated behaviors. Parallel with the blockade of the two transcription factors, amphetamine-stimulated mRNA expression of neuropeptides, preprodynorphin, substance P and preproenkephalin, in the striatum was also reduced by MCPG. MCPG itself, however, had little or no effect on constitutive expression of these genes in the striatum and spontaneous behavioral activity. These data indicate a preferential participation of MCPG-sensitive mGluRs in psychostimulant-stimulated gene expression which is believed to be important for neuroplasticity underlying amphetamine dependence. It is notable that this type of glutamate receptor, as the modulatory receptor in nature, has little impact on fast signal transmission in synapse. Thus, mGluR antagonist, as opposed to ionotropic glutamate receptor antagonists, should cause no major general depression or cognitive side effect in chronic therapy. Along with rapid progress in improving brain penetration and subtype specificity, mGluR agents might serve better therapeutic drugs for substance dependence and addiction. (Supported by NIDA/NIH DA10355 and a Young Investigator Award from NC Governor's Institute and a research grant (4150486810) from Latham Trust of Crestar Bank.)

Licostinel: Potential Solutions to Safety Concerns (GI-A)

Weihmuller, F.

Licostinel (ACEA 1021) is a potent competitive antagonist of the N-methyl-D-aspartate (NMDA) receptor at the glycine site. The strength of safety and efficacy data in animal models has supported the clinical investigation of licostinel for the treatment of ischemic stroke. After obtaining clinical safety data during short (15 min.) infusions of licostinel in 64 stroke patients and 62 healthy volunteers, further investigation of prolonged continuous infusion of licostinel showed formation of crystals in the urine of some subjects. Studies in animal models have shown that the plasma concentration of licostinel at which crystals form in the urine of human subjects was greater (9 times) than the minimum effective plasma concentration (2 ug/ml) required for neuroprotection in stroke models. Furthermore, animal studies have demonstrated that pretreatment with the uricosuric agent probenecid reduces renal excretion and raises plasma levels of licostinel. These findings suggest that a broad margin of safety exists between the minimum efficacious dose and dose-limiting safety concerns. These studies and their implications for future clinical development will be discussed.

Glutamate Receptors Regulate the Convulsant Effects of Cocaine (GI-B)

Witkin, J.M. and Gasior, M.

Convulsions associated with cocaine abuse can be life-threatening and resistant to treatment. Status epilepticus following cocaine poisoning is often resistant to standard therapy and can be fatal. In some models of cocaine seizures, even high, incapacitating doses of anticonvulsant drug standards are ineffective against cocaine convulsions. In contrast, functional antagonists of glutamatergic transmission protect against the toxic consequences of cocaine overdose. NMDA receptor antagonists dose-dependently prevent the clonic convulsions induced by cocaine in rodent models. The role of NMDA receptors in the control of cocaine-induced convulsive activity is strengthened by the finding that anticonvulsant activity can be achieved by blockade of both competitive and uncompetitive modulatory sites on the NMDA receptor complex. Thus, competitive antagonists, ion-channel blockers, polyamine antagonists, and functional blockers of the strychnine-insensitive glycine modulatory site of the NMDA receptor complex all prevent cocaine seizures. Although some NMDA blockers produce profound side-effects, significantly reducing therapeutic indices, others (e.g., low-affinity channel blockers, glycine antagonists) demonstrate significant separation in their anticonvulsant and side-effect profiles. The anticonvulsant efficacy of these compounds can be augmented by diazepam suggesting the potential clinical utility of such drug combinations. Interestingly, functional NMDA antagonists may also be of value in the treatment of cocaine dependence.

Participants

Speaker List

Gary J. Bennett, Ph.D.
Professor
Department of Neurology
Allegheny University of the Health Sciences
Broad and Vine Street (Mail Stop 423)
Philadelphia, PA 19102
(215) 762-1319
(215) 762-3161 fax
bennettg@allegheny.edu

Linda Brady, Ph.D.
Acting Chief
Preclinical and Clinical Therapeutics Research Branch
National Institute of Mental Health
National Institutes of Health
6001 Executive Blvd
Bethesda, Maryland 20892 (301) 443-9875
(301) 443-4822 fax
lbrady@helix.nih.gov

Dennis W. Choi, M.D., Ph.D.
Professor and Head
Department of Neurology
Washington University School of Medicine
Box 8111
660 South Euclid Avenue
St. Louis, MO 63110
(314) 362-7175
(314) 362-1771 fax
choid@neuro.wustl.edu

Enoch Gordis, M.D.
Director
National Institute on Alcohol Abuse and Alcoholism
National Institutes of Health
Willco Building, Suite 400
6000 Executive Boulevard, MSC 7003
Bethesda, MD 20892-7003
(301) 443-3885
(301) 443-7043 fax

J. Timothy Greenamyre, M.D., Ph.D.
Professor
Department of Neurology
Emory University
Woodruff Memorial Research Building, Suite 6000
1639 Pierce Drive
Atlanta, GA 30322
(404) 727-4550
(404) 727-3157 fax
jtg@neuro.emory.edu

Stephen F. Heinemann, Ph.D.
Professor
Molecular Neurobiology Laboratory
The Salk Institute
10010 North Torrey Pines Road
La Jolla, CA 92037
(619) 453-4100, ext. 1112
(619) 552-3469 fax
heinemann@salk.edu

Barbara H. Herman, Ph.D.
Chair, NIH Glutamate Cascade Meeting
National Institute on Drug Abuse
National Institutes of Health
6001 Executive Blvd
Bethesda, Maryland 20892 (301) 443-9799
(301) 443-2599 fax
bh78q@nih.gov

Barry J. Hoffer, M.D., Ph.D.
Scientific Director
National Institute on Drug Abuse
National Institutes of Health
5500 Nathan Shock Drive
Baltimore, MD 21224
(410) 550-1539
(410) 550-1645 fax

Paula L. Hoffman, Ph.D.
Professor
Department of Pharmacology
University of Colorado Health Sciences Center
4200 East Ninth Avenue, C-236
Denver, CO 80262
(303) 315-5690
(303) 315-7097 fax
paula.hoffman@uchsc.edu

Frances E. Jensen, M.D.
Assistant Professor
Department of Neurology
Harvard University School of Medicine
Children's Hospital
Enders 2, Room 249
300 Longwood Avenue
Boston, MA 02115
(617) 355-8439
(617) 738-1542 fax
jensen@a1.tch.harvard.edu

Stuart A. Lipton, M.D., Ph.D.
Chief
CNS Research Institute
Brigham and Women's Hospital
Harvard Medical School
LMRC First Floor
221 Longwood Avenue
Boston, MA 02115
(617) 278-0363
(617) 264-5277 fax
slipton@rics.bwh.harvard.edu

Mitchell B. Max, M.D.
Chief
Clinical Trials Unit
Pain and Neurosensory Mechanisms Branch
National Institute of Dental Research
National Institutes of Health
Building 10, Room 3C-405
9000 Rockville Pike
Bethesda, MD 20892-1258
(301) 496-5483, ext. 405
(301) 402-4347 fax
mm77k@nih.gov

Tracy K. McIntosh, Ph.D.
Professor
Department of Neurosurgery
University of Pennsylvania
3320 Smith Walk, Suite 105
Philadelphia, PA 19104
(215) 573-3156
(215) 573-3808 fax
mcintosh@eniac.seas.upenn.edu

John H. Morrison, Ph.D.
Professor and Director
Neurobiology of Aging Laboratories
Co-Director
Fishberg Research Center for Neurobiology
Mount Sinai School of Medicine
Box 1639
1 Gustave L. Levy Place
New York, NY 10029-6574
(212) 241-1758
(212) 849-2510 fax
morrison@cortex.neuro.mssm.edu

Gavril W. Pasternak, M.D., Ph.D.
Professor
Departments of Neurology, Neuroscience,
and Pharmacology
Cornell University Medical College
Member, Memorial Sloan-Kettering Cancer Center
1275 York Avenue
New York, NY 10021
(212) 639-7046
(212) 794-4332 fax
pasterng@mskmail.mskcc.org

Audrey Penn, M.D.
Acting Director
National Institute of Neurological Disorders and Stroke
National Institutes of Health
Building 31, Room 8A-52
9000 Rockville Pike
Bethesda, MD 20892
(301) 496-3167
(301) 496-0296 fax

Piotr Popik, M.D., Ph.D.
Research Scientist and Chief
Behavioral Division
Department of Biochemistry
Institute of Pharmacology
Polish Academy of Sciences
12 Smetna Street
31-343 Krakow, Poland
1-48-12 6374630
1-48-12 6374500 fax
nfpopik@cyf-kr.edu.pl

Luigi Pulvirenti, M.D.
Assistant Professor
Division of Pharmacology
Department of Biology
University of Rome - Tor Vergata
00133 Rome, Italy
39-6-7259-4875
39-6-2022-720 fax
luigip@tin.it

Michael A. Rogawski, M.D., Ph.D.
Chief
Neuronal Excitability Section
Epilepsy Research Branch
National Institute of Neurological Disorders and Stroke
National Institutes of Health
Building 10, Room 5N-250
10 Center Drive, MSC 1408
Bethesda, MD 20892-1408
(301) 496-8013
(301) 402-6788 fax
rogawski@nih.gov

Philip H. Sheridan, M.D.
Chief
Epilepsy Research Branch
National Institute of Neurological Disorders and Stroke
National Institutes of Health
Federal Building, Room 516
7550 Wisconsin Avenue
Bethesda, MD 20892-9020
(301) 496-6691
(301) 480-2424 fax
ps59i@nih.gov

Phil Skolnick, Ph.D.
Lilly Research Fellow
Neuroscience Discovery
Lilly Research Laboratories
Lilly Corporate Center
Building 48, Drop Code 0510
Indianapolis, IN 46285
(317) 277-9203
(317) 276-7600 fax
skolnick_phil@lilly.com

Bradley Wise, Ph.D.
Director, Fundamental Neuroscience
Neuroscience and Neuropsychology of Aging Program
National Institute on Aging
National Institutes of Health
Gateway Building, Room 3C-307
7201 Wisconsin Avenue
Bethesda, MD 20892
(301) 496-9350
(301) 496-1494 fax
bw86y@nih.gov

Marina E. Wolf, Ph.D.
Associate Professor
Department of Neuroscience
The Chicago Medical School
3333 Green Bay Road
North Chicago, IL 60064-3095
(847) 578-8659
(847) 578-3428 fax
wolfm@mis.finchcms.edu

Walter Zieglgänsberger, M.D., Ph.D.
Head of Clinical Neuropharmacology
Department of Clinical Neuropharmacology
Max Planck - Institute of Psychiatry
Kraepelinstr. 2
D-80804 Munchen, Germany
49-89-30622-350
49-89-30622-402 fax
wzg@mpipsykl.mpg.de

Stephen R. Zukin, M.D.
Director
Division of Clinical and Services Research
National Institute on Drug Abuse
National Institutes of Health
6001 Executive Blvd
Bethesda, Maryland 20892 (301) 443-6697
(301) 443-2317 fax
szukin@helix.nih.gov

Poster Presenter List

Walid M. Abi-Saab, M.D.
Assistant Professor
Department of Psychiatry
Clinical Neuroscience Research Unit
Connecticut Mental Health Center
Yale University School of Medicine
34 Park Street, Room 336B
New Haven, CT 06519
(203) 867-6030
(203) 789-7651 fax
walid.abi-saab@yale.edu

Amit Anand, M.D.
Department of Psychiatry
Yale University School of Medicine
VA Connecticut Healthcare System
Psychiatry Services 116A
950 Campbell Avenue
West Haven, CT 06516
(203) 932-5711, ext. 2546
(203) 937-3886 fax
amit.anand@yale.edu

Michael H. Baumann, Ph.D.
Intramural Research Program
Clinical Psychopharmacology Section
National Institute on Drug Abuse
National Institutes of Health
PO Box 5180
5500 Nathan Shock Drive
Baltimore, MD 21224
(410) 550-1598
(410) 550-2997 fax
mbaumann@intra.nida.nih.gov

Robert M. Berman, M.D.
Abraham Ribicoff Research Center of the Connecticut Mental Health Center
VA Connecticut Healthcare System, West Haven Campus
and Assistant Professor
Department of Psychiatry
Yale University School of Medicine
Clinical Neuroscience Research Unit, Room 360
34 Park Street
New Haven, CT 06519
(203) 789-7329
(203) 789-7651 fax
robert.berman@yale.edu

Willoughby Britton
Research Technician
Integrative Neuroscience Unit
Behavioral Neuroscience Branch
National Institute on Drug Abuse
National Institutes of Health
5500 Nathan Shock Drive
Baltimore, MD 21224
(410) 550-2471
(410) 550-1692 fax
wbritton@intra.nida.nih.gov

De-Maw Chuang, Ph.D.
Chief
Section on Molecular Neurobiology
Biological Psychiatry Branch
National Institute of Mental Health
National Institutes of Health
Building 10, Room 3N-212
10 Center Drive, MSC 1272
Bethesda, MD 20892-1272
(301) 496-4915
(301) 480-9290 fax
chuang@helix.nih.gov

Eain M. Cornford, Ph.D.
Lab Chief
Neurology Department
West Los Angeles VA Medical Center
Neurology W127B
11301 Wilshire Boulevard
Los Angeles, CA 90073
(310) 268-4426
(310) 478-4538 fax
cornford@ucla.edu

James W. Cornish, M.D.
Assistant Professor
Department of Psychiatry
Treatment Research Center
University of Pennsylvania
School of Medicine
3900 Chestnut Street
Philadelphia, PA 19104-6178
(215) 222-3200, ext. 118
(215) 386-6770 fax
cornish@research.trc.upenn.edu

Michael D.R. Croning, D.Phil.
Section of Respiratory Medicine
Department of Pediatrics
Yale University School of Medicine
333 Cedar Street
New Haven, CT 06520
(203) 785-6793
(203) 785-6337 fax
michael.croning@yale.edu

D. Cyril D'Souza, M.D.
Department of Psychiatry
Yale University School of Medicine
VA Connecticut Healthcare System
Psychiatry Service 116A
950 Campbell Avenue
West Haven, CT 06516
(203) 932-5711, ext. 2594
(203) 937-4860 fax
d_souza.deepak_c@west-haven.va.gov or dds020963@pol.net

Denise A. Figlewicz, Ph.D.
Associate Professor
Departments of Neurology and Neurobiology and Anatomy
University of Rochester Medical Center
Box 673
601 Elmwood Avenue
Rochester, NY 14642
(716) 275-4055
(716) 256-1423 fax
dfiglewicz@mail.neurology.rochester.edu

Michael L. Garcia
Mayo Clinic and Mayo Foundation Molecular Neuroscience Ph.D. Program
Mayo Graduate School
1501 Guggenheim Building
200 First Street, SW
Rochester, MN 55905
(507) 284-1781
(507) 284-3383 fax
garcia.michael@mayo.edu

Stacie D. Grossman
Department of Cell Biology
Georgetown University Medical Center
3900 Reservoir Road, NW
Washington, DC 20007
(202) 687-0226
(202) 687-1823 fax
grossmas@medlib.georgetown.edu

Patrick Harty, Ph.D.
IRTA Fellow
Neuronal Excitability Section
Epilepsy Research Branch
National Institute of Neurological Disorders and Stroke
National Institutes of Health
Building 10, Room 5N-250
10 Center Drive
Bethesda, MD 20892-1408
(301) 496-0957
(301) 402-2871 fax
tpatrick@helix.nih.gov

Luc Jasmin, M.D., Ph.D.
Assistant Professor
Department of Neurosurgery
Georgetown University
Research Building, Room W221
3970 Reservoir Road, NW
Washington, DC 20007
(202) 687-0228 or 0229
(202) 687-0341 fax
ljasmi01@gumedlib.dml.georgetown.edu

N. Bradley Keele, Ph.D.
PRAT Fellow
Neuronal Excitability Section
Epilepsy Research Branch
National Institute of Neurological Disorders and Stroke
National Institutes of Health
Building 10, Room 5N-250
10 Center Drive
Bethesda, MD 20892-1408
(301) 496-0957
(301) 402-2871 fax
nbkeele@helix.nih.gov

Jeong-Hoon Kim, Ph.D.
Research Associate
Department of Psychiatry
The University of Chicago
5841 South Maryland Avenue, MC3077
Chicago, IL 60637
(773) 702-2890 or 2891
(773) 702-0857 fax
jeongkim@midway.uchicago.edu

Rebecca C. Klein
Research Assistant
Department of Chemistry and Biochemistry
University of Notre Dame
219 Nieuwland Science Hall
Notre Dame, IN 46556
(219) 631-7733
(219) 631-4048 fax
rklein1@darwin.helios.nd.edu

John H. Krystal, M.D.
Associate Professor
Department of Psychiatry
Yale University School of Medicine
Director
Division of Cognitive and Clinical Neuroscience
VA Connecticut Healthcare System
Psychiatry Service 116A
950 Campbell Avenue
West Haven, CT 06516
(203) 937-4790
(203) 937-3468 fax
john.krystal@yale.edu

He Li, M.D., Ph.D.
Postdoctoral Fellow
Neuronal Excitability Section
Epilepsy Research Branch
National Institute of Neurological Disorders and Stroke
National Institutes of Health
Building 10, Room 5N-250
10 Center Drive
Bethesda, MD 20892-1408
(301) 594-7038
(301) 402-2871 fax
heli@helix.nih.gov

Jinhong Li, M.D.
Graduate Student
Department of Physiology
Georgetown University Medical School
3900 Reservoir Road, NW
Washington, DC 20007
(202) 687-1567
(202) 687-7407 fax
lij@medlib.georgetown.edu

Erich Lieth, Ph.D.
Associate Professor
Department of Neuroscience and Anatomy
The Pennsylvania State University
College of Medicine
M.S. Hershey Medical Center
500 University Drive
Hershey, PA 17033
(717) 531-4104
(717) 531-5184 fax
Lieth@psu.edu

Xianrong Mao
Student
University of Arkansas for Medical Sciences
4301 West Markham Street, Slot 510
Little Rock, AR 72205
(501) 661-1202, ext. 2976
(501) 671-2524 fax
xmao@life.uams.edu

Rae R. Matsumoto, Ph.D.
Assistant Professor
Department of Pharmacology and Toxicology
College of Pharmacy
University of Oklahoma Health Sciences Center
PO Box 26901
Oklahoma City, OK 73190
(405) 271-6593, ext. 47250
(405) 271-7477 fax
rae-matsumoto@uokhsc.edu

Jim L. Morris, Ph.D.
National Research Council Research Associate
U.S. Army Medical Research Institute for Chemical Defense
3100 Ricketts Point Road
Aberdeen Proving Ground, MD 21010-5425
(410) 671-1954
(410) 671-1960 fax
Dr._Jim_morris@ftdetrck-ccmail.army.mil

Marina Nakic, M.D., Ph.D.
ETH - Hoenggerberg, Zuerich, Switzerland
NALJESKOVICEVA 35
10000 ZAGREB - CROATIA
011- (385)-1-534-556
011- (385) - 4826-053 fax
uniondrvo@zg.tel.hr

Gerald Rameau, Ph.D.
Howard Hughes Medical Institute
New York University Medical Center
550 First Avenue
New York, NY 10016
(212) 263-5939
(212) 683-8453 fax
rameag01@mcrcr6.med.nyu.edu

Marc I. Rosen, M.D.
Department of Psychiatry
Yale University School of Medicine
VA Connecticut Healthcare System
Psychiatry Service 116A
950 Campbell Avenue
West Haven, CT 06516
(203) 932-5711, ext. 2112
(203) 937-4937 fax
marc.rosen@yale.edu

Yutaka Sagara, Ph.D.
Research Associate
Cellular Neurobiology Laboratory
The Salk Institute for Biological Studies
10010 North Torrey Pines Road
La Jolla, CA 92037
(619) 453-4100, ext. 1549
(619) 535-9062 fax
sagara@salk.edu

Frank M. Scalzo, Ph.D.
Associate Professor
Department of Pediatrics
Arkansas Children's Hospital Research Institute
800 Marshall Street, MS-512-20B
Little Rock, AR 72202
(501) 320-2747
(501) 320-3161 fax
fmscalzo@life.uams.edu

Ilsa R. Schwartz, Ph.D.
Professor of Surgery/Otolaryngology and Neurobiology
Yale University School of Medicine
PO Box 208041
333 Cedar Street
New Haven, CT 06520-8041
(203) 785-6329
(203) 737-2245 fax
ilsa.schwartz@yale.edu

Yoland Smith, Ph.D.
Yerkes Regional Primate Research Center
and Associate Professor
Department of Neurology
Emory University
954 Gatewood Road, NE
Atlanta, GA 30322
(404) 727 7519
(404) 727 3278 fax
yolands@rmy.emory.edu

Steven Sparenborg, Ph.D.
Health Scientist Administrator
Pharmacology and Toxicology Branch
Medications Development Division
National Institute on Drug Abuse
National Institutes of Health
6001 Executive Blvd
Bethesda, Maryland 20892 (301) 443-8422
(301) 443-9649 fax
ss292q@nih.gov

Michael Sturgess, Ph.D.
Group Leader - Chemistry
Bearsden Bio, Inc.
34 Mt. Pleasant Drive
Aston, PA 14014
(610) 361-4251
(610) 361-8256 fax
sturgess@netreach.net

Masako Tanaka, M.D.
Special Volunteer
Pain and Neurosensory Mechanisms Branch
National Institute of Dental Research
National Institutes of Health
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49 Convent Drive, MSC 4410
Bethesda, MD 20892-4410
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(301) 402-0667 fax
mtanaka@yoda.nidr.nih.gov

John Q. Wang, M.D., Ph.D.
Assistant Professor
Department of Pharmacology
Howard University College of Medicine
520 W Street, NW
Washington, DC 20059
(202) 806-9768
(202) 806-4453 fax
jwang@hucmphrm.howard.edu

Fred Weihmuller, Ph.D.
Senior Project Manager
CoCensys, Inc.
201 Technology Drive
Irvine, CA 92618
(714) 753-6118
(714) 753-6147 fax
fweihmuller@cocensys.com

Jeff Witkin, Ph.D.
Research Psychologist
Drug Development Group
Addiction Research Center
National Institute on Drug Abuse
National Institutes of Health
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(410) 550-1586
(410) 550-1648 fax
jwitkin@intra.nida.nih.gov